J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 DOI 10.1007/s00775-007-0256-4

2. METALLOPROTEINS DNA; the properties of two of these (hABH1 and hABH5) are described. KEYNOTE LECTURES KL10 KL08 Towards the Mechanism of N2 Reduction by Nitrogenase Electrocatalytic hydrogen cycling by hydrogenases in the Brian M. Hoffman, Northwestern University, Evanston, IL, USA. Contact e-mail: [email protected] presence of O2: functional challenges for technology Fraser Armstrong1, Kylie A. Vincent1, James A. Cracknell1, 1 1 2 A major obstacle to understanding the reduction of N2 to NH3 by Annemarie Wait , Gabrielle Goldet , Baebel Friedrich , Oliver nitrogenase has been the impossibility of synchronizing electron Lenz2, Marcus Ludwig3, 1University of Oxford, Department of 2 delivery to the MoFe protein so that intermediates along the N2 Chemistry, Oxford, United Kingdom; Institut für reduction pathway can be accumulated for characterization. Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Germany; 3 Recently, however, in a collaboration with the groups of Dennis Institut für Biologie/Mikrobiologie,, Humboldt-Universität zu Dean and Lance Seefeldt, a number of intermediates have been Berlin, Germany. trapped by freeze-quenching, and ENDOR spectroscopy has proven Contact e-mail: [email protected] to be the method of choice for characterizing substrate-derived species bound to FeMo-co of trapped enzymatic intermediates. This Hydrogenases are usually inactivated under oxidizing conditions presentation will describe current understanding of the structure of [1]. Yet many aerobic microbes obtain energy using [NiFe]- trapped intermediates and of the mechanistic sequence of hydrogen hydrogenases that can oxidize traces of H in air [2,3]. Enzymes 2 addition to N2. from Ralstonia have such high selectivity for H against O that 2 2 Even when an intermediate is trapped, the process of electron they can be used as electrocatalysts in the simplest of fuel cells- delivery requires that this occur without synchronous electron lacking a membrane or even just an anode and cathode separated by delivery, and as a result the number of electrons (and protons), n, an electrolyte film [4,5]. Using a ‘blue’ Cu oxidase, laccase as that has been accumulated during its formation is unknown. cathode catalyst, sufficient power is produced from 3% H in air to 2 Consequently, the intermediate is untethered from Lowe-Thornely power a wristwatch - not a high demand, but a visible and (LT) kinetic schemes for reduction, which are indexed by n. We demanding test of a hydrogenase’s ability to function in air. This have shown that a trapped intermediate itself provides a lecture addresses how these hydrogenases function in low levels of 'synchronously prepared' initial state whose relaxation to the resting H . 2 state under conditions that prevent electron delivery to MoFe 1. K. A. Vincent, A. Parkin, O. Lenz, S. P. J. Albracht, J. C. protein can be analyzed to disclose n and the nature of its relaxation Fontecilla-Camps, R. Cammack, B. Friedrich and F. A. Armstrong, reactions. We will describe the relaxation protocol and outline J. Am. Chem. Soc. (2005) 127, 18179. current understanding of the connections of trapped nitrogenous 2. T. Burgdorf, O. Lenz, T. Buhrke, E. van der Linden, A.K. Jones, intermediates to the LT kinetic scheme for N2 reduction, as well as S.P.J. Albracht and B. Friedrich, J. Mol. Microbiol. Biotech. (2005) the mechanisms by which intermediates relax. 10, 181. 3. K. Knuttel, K. Schneider, A. Erkens, W. Plass, A. Muller, E. Bill and A.X. Trautwein, Bull. Polish Acad. Sci.-Chem. (1994) 42, 495. KL11 4. K. A. Vincent, J. A. Cracknell, O. Lenz, B. Friedrich and F. A. Armstrong, Proc. Natl Acad. Sci. USA, (2005) 102, 16951-16954. Copper Prion Interactions. Specificity from mammalians 5. K. A. Vincent, J. A. Cracknell, J. Clark, O. Lenz, M. Ludwig, B. to Fishes 1 1 1 Friedrich and F. A. Armstrong, Chem. Commun. (2006) 5033-5034. Henryk Kozáowski , Anna Janicka , Paweá StaĔczak , Daniela Valensin2, Gianni Valensin2, 1Faculty of Chemistry, University of Wroclaw, Wrocáaw, Poland; 2Department of Chemistry, University KL09 of Siena, Siena, Italy. Fe(II)/2-oxoglutarate-dependent hydroxylases Contact e-mail: [email protected] Robert P. Hausinger1, Piotr K. Grzyska1, Meng Li2, Jana M. Prion diseases are fatal neurodegenerative disorders including Simmons3, Tina A. Müller1, 1Michigan State University, spongiform encephalopathies in cattle and sheeps and Creutzfeld- Microbiology and Molecular Genetics, East Lansing, MI, USA; Jacob syndrome in humans. Although biological functioning of 2Michigan State University, Chemistry, East Lansing, MI, USA; prion protein (PrP) is still unknown it seems to be generally 3Michigan State University, Biochemistry and Molecular Biology, accepted that it may play a critical role in copper homeostasis and East Lansing, MI, USA. copper based enzymatic activity.1 Contact e-mail: [email protected] Mammalian protein possesses two specific binding sites in the unstructured domain: i) octarepeat region2 and ii) neurotoxic 2-oxoglutarate-dependent hydroxylases are mononuclear non-heme peptide fragment.3 In all cases histidine residues play a basic role in Fe(II) enzymes that couple the oxidative decarboxylation of an oxo- the metal protein interactions. Avian proteins contain the acid to the transformation of a primary substrate. The archetype hexapeptide repeats having also one His residue critical for Cu(II) representative of this family is TauD, an binding.4 Surprisingly the fish proteins e.g. that of Japanese enzyme that converts taurine (2-aminoethanesulfonate) to sulfite pufferfish may be even more potent Cu(II) binder than those of and aminoacetaldehyde. Recent spectroscopic findings related to mammalians and avian.5 In the latter case the repeat domain is very TauD and its variants will be presented and corresponding insights different than those in human or chicken, but containing multi- into the enzyme mechanism will be described. To illustrate the histidine region is very effective in the interactions with copper. versatility of this group of catalysts, three representative enzymes 1. E. Gaggelli, H. Kozlowski, D. Valensin, and G. Valensin, Chem. from eukaryotic sources will be discussed. Aspergillus nidulans Rev., 106, 1995, 2006. contains XanA which was demonstrated to be a xanthine 2. D. Valensin, M. Luczkowski, et al., Dalton Trans., 1284, 2004. hydroxylase; this enzyme utilizes 2-oxoglutarate-dependent 3. F. Berti, E. Gaggelli, R. Guerrini, A. Janicka, et al., Chem. Eur. J. chemistry rather than the better known molybdopterin system for 13, 1991, 2007. producing uric acid. Trypanosoma brucei, the protozoan responsible 4. P. Stanczak, D. Valensin, et al., Chem. Commun., 3298, 2005. for African sleeping sickness, is known to modify specific thymine 5. P. Stanczak, D. Valensin, et al., Biochemistry, 45, 12227, 2006. bases in its DNA to create the J base; evidence is presented related to the potential use of a 2-oxoglutarate-dependent thymine hydroxylase catalyzing this activity. Finally, humans possess eight homologues to AlkB, an enzyme that repairs alkylation-damaged 123 S54 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

KL12 Living on acetylene: structure and function of acetylene hydratase, a novel bacterial tungsten enzyme Grazyna Seiffert1, Felix TenBrink1, Bernhard Schink1, Albrecht Messerschmidt2, Oliver Einsle3, Peter M. H. Kroneck1, 1Universität Konstanz, Konstanz, Germany; 2MPI Biochemie, Martinsried, Germany; 3Universität Göttingen, Göttingen, Germany. Contact e-mail: [email protected]

To date, acetylene is the only hydrocarbon known to be metabolized in the absence and presence of molecular oxygen. The novel W,Fe- S enzyme acetylene hydratase (AH) from the strictly anaerobic bacterium P. acetylenicus stands out from its class in that it catalyzes a non-redox reaction, the addition of water to the C,C triple bond to form acetaldehyde. However, Ti(III)-citrate or dithionite, is required for activity. AH belongs to the dimethylsulfoxide reductase family, and it contains a bis-MGD- ligated W and a [4Fe:4S] cluster, with a redox potential of - 410 ± 20 mV [1]. The 3D structure (1.26 Å) reveals a water molecule at the W site that gets activated by an aspartate to attack acetylene bound in a hydrophobic pocket. A strong shift in pKa of the aspartate residue is required, caused by the [4Fe:4S] cluster. To KL14 access this novel W site, AH evolved a substrate channel distant The molybdenum : Biosynthesis, function and from where it is found in related Mo and W enzymes [2]. P. deficiency acetylenicus can also insert Mo (but not V) into the bis-MGD Guenter Schwarz, Institute of Biochemistry at the University cofactor of AH, but the specific activity of the Mo isoenzyme was of Cologne, Cologne, Germany. significantly lower. Contact e-mail: [email protected] [1] Boll, M., Schink, B., Messerschmidt, A., Kroneck, P.M.H., Novel bacterial molybdenum and tungsten enzymes: Three- The molybdenum cofactor (Moco) forms the of all dimensional structure, spectroscopy, and reaction mechanism, Biol. molybdenum enzymes, except nitrogenase. Molybdenum enzymes Chem., 2005, 386, 999-1006. catalyze important redox reactions in global metabolic cycles. Moco [2] Seiffert, G.B., Ullmann, G.M., Messerschmidt, A., Schink, B., consists of molybdenum covalently bound to one or two dithiolates Kroneck, P.M.H., Einsle, O. Structure of the non-redox-active attached to a unique tricyclic pterin moiety commonly referred to as tungsten/[4Fe-4S] enzyme acetylene hydratase, Proc. Natl. Acad. molybdopterin (MPT). Moco is synthesized by an ancient and Sci. (USA), 2007, 104, 3073-3077. conserved biosynthetic pathway that can be divided into four major steps, according to the biosynthetic intermediates precursor Z (cyclic pyranopterin monophosphate), MPT, and adenylated MPT. KL13 Before molybdenum is inserted, a Cu(I) atom is bound via the MPT Discriminating Heavy Metal Binding to De Novo dithiolene sulfurs. In a fifth step modifications such as attachment designed peptides using spectroscopy and x-ray of nucleotides, sulfuration, or bond formation between crystallography Molybdenum and the protein result in different catalytic centers Vincent L. Pecoraro1,2, Olga Iranzo1, Debra Touw1, Christopher formed by Moco. Upon completion of cofactor biosynthesis Moco Cabello1, Kyung-Hoon Lee1, Saumen Chakravorty1, 1University of can be bound to a carrier protein. A defect in any of the steps of Michigan, Chemistry, Ann Arbor, MI, USA; 2University of Moco biosynthesis results in the pleiotropic loss of all molybdenum Michigan, Biophysics Research Division, Ann Arbor, MI, USA. enzyme activities, causing the death of the organism. Human Moco Contact e-mail: [email protected] deficiency is a hereditary metabolic disorder characterized by severe neurodegeneration resulting in early childhood death. Latest The ability to control the coordination properties of metals (number developments in the field of bacterial and eukarytic Moco of ligands, geometry, etc.) in proteins is an essential feature for the biosynthesis will be presented. Finally, novel insights into the design of metalloenzymes. We will discuss the factors that allow us biosynthesis of related tungsten-containing pterin cofactors will be to differentiate the relative binding affinities of the same metal for discussed. apparently identical sites within moderately long peptides and discriminate between different metals for chemically identical sites. Using the designed Į-helical 3-stranded coiled coil peptides known KL15 as the TRI peptides (sequence G-(LKALEEK)4G in which one or Methyl-coenzyme M reductase and the anaerobic two of the leucines (L) is substituted by cysteine (C)) we will oxidation of methane describe the protein chemistry of toxic heavy metals such as Pb(II), Rudolf K. Thauer, Max-Planck-Institut für terrestrische As(III), Hg(II) and Cd(II) with thiolate rich protein sequences. We Mikrobiologie, Marburg/Lahn, Germany. will show that Cd(II) can be controlled to bind as either a three or Contact e-mail: [email protected] four coordinate structure in aqueous solution and we will show that individual peptides can be made that contain two 4-coordinate, two Methane has long been known to be used as carbon and energy 3-coordinate or a mixed 3 and 4 coordinate peptides. We will also source by some aerobic alpha- and delta-proteobacteria. In these discuss site selective metal binding where Cd(II) can be forced to organisms the metabolism of methane starts with its oxidation with one site in this protein to the exclusion of a second identical binding O2 to methanol, a reaction catalyzed by a monooxygenase and site and when simultaneously challenged with both Pb(II) and therefore restricted to the aerobic world. Methane has recently been Cd(II), one can selectively sequester metals into binding sites that shown to also fuel the growth of anaerobic microorganism. The again appear identical. This selectivity will be discussed using oxidation of methane with sulfate and with have been crystallographic studies of an As(III) peptide. reported, but the mechanisms of anaerobic methane oxidation still remains elusive. Sulfate-dependent methane oxidation is catalyzed by methanotrophic archaea, which are related to the Methanosarcinales and which grow in close association with sulfate 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S55

reducing delta-proteobacteria. There is evidence that anaerobic hydrogen bond between propionic acid and Arg177 of HO. Due to methane oxidation with sulfate proceeds at least in part via reversed the hydrogen bonding formation, the ET rate from NADPH/CPR to methanogenesis involving the nickel enzyme methyl-coenzyme M the composite is 3.5-fold faster than that of Fe(Schiff-base)/HO, reductase for methane activation.. though a redox potential of Fe(carboxylate-Schiff-base)/HO (-79 Krüger, M., Meyerdierks, A., Glöckner, F. O., Amann, R., Widdel, mV vs. NHE) is lower than that of Fe(Schiff-base)/HO (+15 mV vs. F., Kube, M., Reinhard, R., Kahnt, J., Böcher, R., Thauer, R. K., & NHE). Instead of these small protein cavities, we have also Shima, S. (2003) A conspicuous nickel protein in microbial mats employed a protein having a large cavity, i.e., ferritin (Fr). We have that oxidize methane anaerobically. Nature 426, 878-881. prepared a zero-valent palladium cluster by chemical reduction of Shima, S. & Thauer, R. K. (2005) Methyl-coenzyme M reductase palladium ions in the apo-ferritin cage and examined its catalytic and the anaerobic oxidation of methane in methanotrophic Archaea. reduction activity. Curr. Opin. Microbiol. 8, 643-648. Thauer, R. K. & Shima, S. (2006) Methane and microbes. Nature 440, 878-879. SESSION LECTURES

SL030 KL16 New fluorescent probes for Cytochrome P450 3A4 Zinc and copper in the brain: Unraveling the role of (CYP3A4) metallothionein-3 Antoinette Chougnet, University of Basel, Department of Milan Vašák, Department of Biochemistry, University of Zürich, Chemistry, Basel, Switzerland. Zürich, Switzerland. Contact e-mail: [email protected] Contact e-mail: [email protected] Steroid derivatives were synthesized carrying fluorescent groups Zinc and copper homeostasis plays a crucial role in brain such as anthracene, dansyl, deazaflavin, and pyrene attached to C-6, physiology and in neurodegenerative diseases. Metallothionein-3 e.g. see 1. These compounds are unique inhibitors of Cytochrome (Zn7MT-3) is mainly expressed in the brain and was found P450 3A4 displaying similar IC50 values in the low micro-M range downregulated in Alzheimer's disease (AD). The protein occurs for the 3A4 substrates midazolam, testosterone and nifedipine. On intra- and extracellularly in comparable amounts. Intracellular binding to 3A4 the fluorescence of the dansyl, deazaflavine or Zn7MT-3 is expressed in glutamatergic neurons that release zinc pyrene probes is quenched due to photophysical interaction of the from their synaptic terminals. We could demonstrate a specific fluorophore with the heme. Addition of drug candidates with nM- binding of Zn7MT-3 to the small GTPase Rab3A-GDP. This ȝM binding constants causes displacement of the probes from the interaction indicates that Zn7MT-3 is not merely a cellular zinc active site and hence leads to restoration of the fluorescence. buffer, but actively participates in synaptic vesicle trafficking Accordingly relative affinities of drug candidates to CYP3A4 can upstream of vesicle fusion. Involvement of aberrant metal-protein be easily and accurately determined by fluorescence measurements interactions and oxidative stress play a key role in AD. The The mechanism of fluorescence quenching was investigated with production of reactive oxygen species (ROS) and neuronal toxicity the synthetic model compound 2 in which deazaflavin is strapped are linked to the binding of redox-active metals like copper to the over the distal side of an iron(III)-porphyrin to mimic the envisaged amyloid-beta peptide (Aȕ). The protective effect of extracellular enzyme-inhibitor interaction within the active site of 3A4, see 1. Zn7MT-3 from Aȕ toxicity in neuronal cell cultures has been Femtosecond pump-probe and fluorescence spectroscopies were demonstrated, but its origin is not understood. By using several used to study the photophysical processes of 2. Accordingly, rapid complementary spectroscopic, biochemical, and cell biological intramolecular energy transfer and enhanced intersystem crossing techniques we could show that Zn7MT-3 not only scavenges free processes induced by the high-spin Fe(III) are responsible for the Cu(II) ions but also removes Cu(II) bound to Aȕ. A metal swapping complete suppression of deazaflavin fluorescence. between Zn7MT-3 and the soluble and aggregated Aȕ-Cu(II) complexes, accompanied by copper reduction, abolishes the ROS production and profoundly reduces the cellular toxicity. The generated Cu(I)4Zn4MT-3 contains an oxygen stable Cu(I)4-thiolate cluster and two disulfide bonds. The results provide insights into the protective role of Zn7MT-3 from Aȕ-Cu(II) toxicity in AD.

KL17 Construction of organometalloproteins: A new approach for bioinorganic chemistry Yoshihito Watanabe, Nagoya University, Grad. Sch. Sci. Dept. Chemistry, Nagoya, Japan. Contact e-mail: [email protected]

Myoglobin (Mb) is a small (17 kDa), well characterized heme protein that is often used as a model system for other heme proteins SL031 and the reactions they catalyze. Very recently, we have designed a mutant, F43W/H64D Mb, in which an aromatic Protein Evolution and Geochemical Changes: From substrate locates immediate above the heme as a model for a Cytochrome c6 to Plastocyanin substrate bound form of cytochrome P450 and site specific aromatic Miguel A. De la Rosa, Instituto de Bioquimica Vegetal y hydroxylation was found to proceed by a stochiometric amount of Fotosintesis, Sevilla, Spain. H2O2 in a few seconds. As an extension of our efforts for the Contact e-mail: [email protected] construction of organometalloproteins, we have replaced the heme prosthetic group with a series of M(salophen) complexes. We have Plastocyanin and cytochrome c6 are two proteins with different also applied this strategy to heme (HO) which is known structures - the former is a cupredoxin, the latter is a heme protein - to catalyze the conversion of heme to biliverdin. In this particular but they can act in a similar way to transfer electrons from the case, we have introduced FeIII(Schiff-base) bearing a carboxylate cytochrome b6-f complex to photosystem I. They are thus an group, instead of heme, located at the active site of HO. The crystal excellent model system for the analysis of biological convergent structure of an Fe(carboxylate-Schiff-base)/HO composite shows a evolution at the molecular level. It seems that cytochrome c6 was 123 S56 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

first synthesized by the most primitive oxygen-evolving (1) Lill R, Mühlenhoff U (2006) Ann Rev Cell Dev Biol 22:457-486 photosynthetic organisms in the early Earth’s life, when iron was (2) Loiseau L et al (2007) Proc. Natl Acad. Sci. (in press) much more available than copper because of the reducing character of the atmosphere. Later on, plastocyanin and other copper proteins appeared as the atmospheric oxygen concentration increased so as SL034 to make the copper element available to living cells. The transition Plant MTs - long neglected members of the from cytochrome c6 to plastocyanin may have indeed favored the metallothionein superfamily evolution of the reaction mechanism with their two membrane Eva Freisinger, Institute of Inorganic Chemistry, University of partners, and electrostatics gained weight over hydrophobic forces Zurich, Zurich, Switzerland. in the binding interactions. The whole reaction mechanism consists Contact e-mail: [email protected] of several steps, including long-range attractive movements, molecular rotations and orientation at short distances, formation of Metallothioneins (MTs) are a class of small, cysteine-rich proteins an encounter complex with a well-defined interaction area and an ubiquitous all across the different kingdoms of life. Characteristic is electron transfer pathway, and electron transfer itself. So such a also the pronounced affinity for metal ions with d10 configuration, functional evolution affected the way these small electron carriers which are bound in form of metal-thiolate clusters using the recognize and bind their membrane partners, changed the step deprotonated thiolate groups of the cysteine residues as ligands. becoming the kinetic bottleneck of the reactions, and provided an Although the discovery of the first plant metallothionein reaches increase in both specificity and efficiency. back more than 20 years, their characterization was paid little attention. This is even more surprising considering that the amino acid sequences and cysteine distribution patterns of plant MTs SL032 differ distinctively from the isoforms of other organisms. Plant MTs Mechanism(s) of the re-oxidation reactions of substrate- are sub-grouped into four families, whose members show a reduced amine oxidases with dioxygen tremendously broader sequence variety than those of the Eric M. Shepherd1, Justine Roth2, David M. Dooley1, 1Montana mammalian subfamilies. Most striking are perhaps the differences State University, Chemistry and Biochemistry, Bozeman, MT, in cysteine content ranging from 10-17 residues per protein, or 15- USA; 2Department of Chemistry, Johns Hopkins University, 20% of total amino acids. The reason why plants display such Baltimore, MD, USA. sequence diversity is largely unknown so far. Contact e-mail: [email protected] A spectroscopic investigation of the most peculiar metal ion binding abilities of a number of plant metallothioneins will be presented, Although the catalytic mechanism of copper-containing amine also considering the redox state of the environment. In addition, I oxidases is reasonably well understood, a critical issue remains to will elaborate shortly on our possibly most peculiar finding so far, be definitively resolved. This is the mechanism by which substrate- the existence of a two-metal ion cluster, called the Ȗ-domain, which reduced enzyme (containing the aminoquinol form of the TPQ was unreported of in the MT superfamily so far [1]. cofactor) is oxidized by molecular oxygen. We have investigated Financial support for this project comes from the Swiss National the reaction of O2 with substrate-reduced amine oxidases by Science Foundation (200020-113728/1 to EF). stopped-flow kinetics, focusing on the amine oxidase from [1] Peroza EA, E. Freisinger E, J. Biol. Inorg. Chem. 2007, DOI Arthrobacter globiformis, and the measurement of 18O isotope 10.1007/s00775-006-0195-5. effects. The effects of temperature, dioxygen concentration, and solvent (D2O versus H2O) have been examined. These data provide additional insight into the reaction of O2 with the [Cu(II) - SL035 aminoquinol] and the [Cu(I) - semiquinone] states of the substrate- Structure and function of the Na+ -translocating NADH reduced amine oxidases examined. dehydrogenase from Vibrio cholerae, a flavo-FeS redox pump Julia Fritz-Steuber, University of Zurich, Zurich, Switzerland. SL033 Contact e-mail: [email protected] Biosynthesis of Iron-Sulfur Clusters: an essential iron- sulfur protein for respiratory metabolism in Escherichia The Na+ -translocating NADH dehydrogenase (Na+ -NQR) is a coli multisubunit respiratory complex containing one 2Fe-2S cluster and Marc Fontecave, Laboratoire de Chimie et Biologie des Métaux; several flavins that couples the oxidation of NADH to the transport UMR 5249 Université Joseph Fourier; CNRS ; CEA ; DSV/iRTSV, of Na+ across the inner bacterial membrane. The enzyme from Grenoble, France. Vibrio cholerae contributes to extracellular superoxide production Contact e-mail: [email protected] which might augment the pathogenicity of the bacterium in the human host (Lin et al, 2007). In our effort to assign electron transfer Several multiprotein complexes assisting Fe-S cluster assembly reactions to individual cofactors in the complex, we studied the have been identified in both procaryotes and eucaryotes (1). redox properties of the individual NqrF subunit which contains one Escherichia. coli contains two biosynthetic machineries named SUF FAD and one [2Fe-2S] cluster. The FAD in subunit NqrF represents and ISC (1). Both contain homologous proteins, SufA and IscA the flavin cofactor with the most negative midpoint potential in the respectively, which have been proposed to function as scaffold Na+-NQR, according to its function as hydride acceptor from proteins, even though this is not firmly established. Scaffold NADH and electron entry site into the complex. From there, proteins provide sites for binding iron and sulphur atoms and electrons are transferred to the FeS cluster in subunit NqrF which transient assembly of clusters which can be subsequently transferred represents the first example for a vertebrate-type [2Fe-2S] cluster in for maturation of Fe-S proteins. We have identified in Escherichia a respiratory complex (Lin et al., 2005). The Na+ -NQR complex coli a new A-type scaffold, named ErpA, which was found essential was overproduced in Vibrio cholerae and purified by affinity for E. coli to grow in the presence of oxygen or alternative electron chromatography. Electron microscopic analysis of the Na+ -NQR acceptors. Defect in isoprenoid biosynthesis was identified as a complex stained with uranyl formate revealed particles with a larger cause of this phenotype. First, the eucaryotic mevalonate-dependent elongated domain (11 x 6 mm) and a smaller globular protrusion pathway for biosynthesis of isopentenyl diphosphate (IPP) with a diameter of 3-4 nm. suppresses the respiratory defects of an erpA mutant. Second, erpA Lin, P.-C., Puhar, A., Türk, K., Piligkos, S., Bill, E., Neese, F., mutant contains greatly reduced amounts of ubi- and mena-quinone. Steuber, J. (2005) J. Biol. Chem., 280, 22560 - 22563 Third, ErpA binds Fe-S clusters and transfers them to apo-IspG, a Lin, P.-C., Türk. K., Häse, C.C., Fritz, G., Steuber, J. (2007) J. protein catalysing IPP biosynthesis in E. coli. ErsA is the first A- Bacteriol., in press type scaffold endowed with a central role in cellular physiology (2). 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S57

SL036 (1) Hirose, J, Fukasawa, K.M. et al. (2001) Biochemistry 40, 11860- The Nature and Function of the Catalytic Centres of the 11865. Arch. Biochem. Molybdenum and Tungsten Oxotransferase Enzymes (2) Hirose, J. , Fukasawa K.M. et al. (2004) Biophys. Dave Garner1, Lisa J. Stewart2, Mark A. Smith3, John A. Joule2, 431, 1-8. Tarnjeet S. Bhachu3, Freya Hine3, E. Stephen Davies3, Ian H. Hillier2, Jonathan P. McNamara2, Helen J. Disley3, Jonathan McMaster3, 1University of Nottingham, School of Chemistry, SL038 Nottingham, United Kingdom; 2The Department of Chemistry, Oxygen Binding Properties of Carcinus aestuarii University of Manchester, Manchester, United Kingdom; 3The Hemocyanin Revealed by Laser Flash Photolysis School of Chemistry, University of Nottingham, Nottingham, Shun Hirota1, Takumi Kawahara2, Mariano Beltramini3, Paolo Di United Kingdom. Muro3, Noriaki Funasaki2, Luigi Bubacco3, 1Nara Institute of Contact e-mail: [email protected] Science and Technology, PRESTO, JST, Nara, Japan; 2Kyoto Pharmaceutical University, Kyoto, Japan; 3University of Padova, Molybdenum and tungsten are the only 4d (Mo) and 5d (W) Padova, Italy. transition metals that are required for the normal metabolism of Contact e-mail: [email protected] biological systems. Mo enzymes are present in bacteria, plants, animals, and humans; their W counterparts occur less frequently. Hemocyanins (Hcs) are cooperative oxygen carrier and storage Virtually all the reactions catalysed by these enzymes involve the proteins in molluscs and arthropods. In the latter, Hc is an net transfer of an oxygen atom either to or from the substrate. The oligomeric protein with a minimal functional subunit of 75 kDa structures of several Mo- and W-enzymes have been determined by designated as 5S. The subunits in Carcinus aestuarii Hc are protein crystallography; in each case the metal is bound to the arranged both as hexamers (16S, 10%) and dodecamers (24S, 90%) 2+ dithiolene group of one (or two) “molybdopterin” (MPT) in the presence of 20 mM Ca at pH 7.5. The O2 binding properties cofactor(s). of Hc show strong pH-dependence (Bohr effect). The structures of We have shown that Rhodobacter capsulatus DMSO reductase is oxy and deoxygenated Limulus polyphemus Hc have been shown to active with either Mo or W as the catalytic centre. This W-DMSO be different from the structure of Panulirus interruptus reductase is the only isoenzyme for which structural, spectroscopic, deoxyhemocyanin (deoxyHc) by X-ray crystallographic studies, and electrochemical, and kinetic characterisation have been achieved the former structures have been proposed to correspond to the low thus allowing for direct comparisons with its Mo counterpart. In affinity (T state) form, whereas the latter structure to the high respect of catalytic activity, W-DMSOR >> Mo-DMSOR for the affinity (R state) form. Although type 3 copper proteins, in reduction of DMSO to DMS; however, unlike Mo-DMSOR, W- particular Hc, have been studied extensively in the bioinorganic DMSOR shows (virtually) no ability to catalyse the oxidation of field because of the unique reversible O2 binding at the dicopper DMS to DMSO. Reasons for the significantly different activity of center, information on the O2-binding character of these proteins is these equivalent Mo and W centres will be presented. The role of limited. We have recently shown that O2 dissociates from D145, a conserved active site residue of Rhodobacter sphaeroides monomeric oxytyrosinase by 355-nm pulse irradiation and rebinds DMSO reductase has been examined.The structure of the MPT is to the produced deoxytyrosinase [1]. In this study, we investigated remarkably conserved in the Mo and W enzymes, but one the O2 binding kinetics of Hc by flash photolysis and found the O2- crystallographic study has shown that the pyran ring can be closed binding properties related to both the Bohr effect and . or open. Possible roles for MPT in the catalysis accomplished by [1] S. Hirota, T. Kawahara, E. Lonardi, E. de Waal, N. Funasaki, G. the Mo and W enzymes will be discussed. Canters, J. Am. Chem. Soc. 127 (2005) 17966-17967.

SL037 SL039 The EPR spectra of the Enzyme-Substrate (Lys-Ala- Molecular Design of Converting an Electron Transfer Naphthylamide) Complex in Cu(II)-Dipeptidyl Peptidase Metalloprotein into a Biocatalyst III Zhong X. Huang, Fudan University, Chemistry, Shanghai, China. Junzo Hirose1, Hiroyuki Iwamoto1, Kayoko Fukasawa2, Contact e-mail: [email protected] 1Fukuyama University, Fukuyama, Japan; 2Matsumoto Dental University, Shiojiri, Japan. Various classes of natural hemoproteins perform a wide variety of Contact e-mail: [email protected] biological activities, such as oxygen carrier, electron transfer, biochemical reaction catalyses, and signal transduction, although they Dipeptidyl peptidase III (DPP III), which has a HELLGH....E share the same protoheme IX as prosthetic group. The artificial design (residues 450-455, 508) motif as the zinc-, is classified of metalloproteins with desired or novel properties and reactivity is as a zinc-aminopeptidase, but the zinc-binding motif (HExxxH (x: one of the challenges in chemical biology research. Hemoproteins are appropriate amino acid)) of DPP III is different from a common the excellent natural models and starting-molecules. zinc-binding motif (HExxH) such as that of thermolysin(1). The In order further to convert an electron transfer hemoprotein into a Cu2+ derivatives of thermolysin and carboxypeptidse A do not have catalytic metalloprotein molecular design based on cytochrome c has enzyme activities. As reported previously(2), the Cu2+ derivative of been employed. Compared to the heme active site of a DPP III has the high enzyme activity and we showed that the motif series single-, double- and triple-site mutant proteins of yeast iso-1- part of DPP III directly influences the expression of the enzyme cytochrome c were generated to eliminate the axial ligand and to activity in the copper derivative of DPP III. For Arg-Arg- introduce a distal histidine and arginine in the heme pocket at residue naphthylamide, the value of kcat/Km of Cu(II)-DPP III was 1/3 that position Met80, Tyr67, Pro71. The obtained three variants, M80V, of Zn(II)-DPP III. Lys-Ala-naphthylamide was also the good Y67H, and Y67H/M80V showed much higher activity substrate for Cu(II)-DPP III but slowly digested at low temperature than the wild-type cytochrome protein. More interestingly, the Cyt c by Cu(II)-DPP III. The EPR spectra of Cu(II)-DPP III mixed with Y67H variant showed the kcat/Km value towards classical guaicol substrate(1-10 mM), Lys-Ala-naphthylamide, at low temperature catalytic reaction not only the highest among the cytochrome c and immediately frozen in liquid nitrogen were measured and were variants but also higher than that of native horseradish peroxidase different from that of Cu(II)-DPP III. The EPR spectra of the under the same condition. A wide range of spectroscopic studies and substrate-Cu(II)-DPP III complexes gradually change to original biocatalytic activity assay provide important insight into the structure- EPR spectrum of Cu(II)-DPP III when the substrate-Cu(II)-DPP III property-reactivity-function relationship of hemoproteins. complexes were incubated at 10 oC. These results clearly indicated This project is supported by the National Science Foundation of that the substrate-Cu(II)-DPP III complex could be measured as China EPR spectrum. 123 S58 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

SL040 SL042 Weak Interaction Regulates the Active Site Structure of a Conformational stability of the CuA center of Blue Copper Protein, Pseudoazurin cytochrome oxidase Takamitsu Kohzuma, Ibaraki University, Institute of Applied Nusrat J. M. Sanghamitra, Shyamalava Mazumdar, Tata Institute Beam Science, Mito, Japan. of Fundamental Research, Department of Chemical Sciences, Contact e-mail: [email protected] Mumbai, India. Contact e-mail: [email protected] Weak non-covalent interactions in biological molecules including proteins, nucleic acids, and lipids play important roles for Cytochrome oxidase forms the terminal step in the respiratory expressing the specific functions and structures in many varieties of electron transfer chain. The dinuclear copper centre (CuA) at the biological systems. subunit II of this enzyme is the electron entry site of the enzyme. Pseudoazurin (PAz) is a blue copper protein, which functions as the The CuA site has been shown to exist in a charge-delocalised form electron carrier in several microorganisms. The aromatic acid or in a valance trapped form, and both of these two forms have been substitutants of PAz, Met16Phe, Met16Tyr, and Met16Trp were proposed to be important for the function of the enzyme. Studies on studied to elucidate the effect of weak indirect interactions the CuA site from various sources have indicated that these two involving pi-pi interaction on the active site structures and forms exist in a pH coupled equilibrium. properties. The present talk will describe some of our recent studies on the Electronic absorption spectra of Met16X variants indicate the stability of the CuA site and the role of proton transfer on the changes in the intensity of the two main LMCT transitions in the stability of the active conformation of the metal center. The results visible region. EPR spectra of Met16X variants at 77 K demonstrate will be discussed in the light of understanding the possible role of that the copper site has two different conformers, characterized by these two forms on the biological electron transfer mediated by the rhombic and axial EPR signals. The EPR spectra indicate that the CuA center. active structure is sufficiently flexible to be modulated by weak interactions between a ligand histidine ring and the newly introduced aromatic rings of Met16Phe, Met16Tyr, and Met16Trp. SL043 The spectroscopic and electrochemical results suggest the second Post-translational assembly of the molybdenum-copper- shell weak interaction may have a role to modulate the structure and sulphur active site cluster of CO dehydrogenase reactivity of blue copper protein active site. Astrid M. Pelzmann1, Marion Ferner1, Holger Dobbek1, Manuel A part of this work is supported by Research Promotion Bureau, Gnida2, Wolfram Meyer-Klaucke2, Ortwin O. Meyer1, 1University Ministry of Education, Culture, Sports, Science and Technology of Bayreuth, Bayreuth, Germany; 2European Molecular Biology (MEXT), Japan to TK, under the contract No. 17-214 and a Grant- Laboratory (EMBL), Outstation Hamburg at DESY, Hamburg, in-Aid for Scientific Research from JSPS (No. 18550147), Japan to Germany. TK. Contact e-mail: [email protected]

CO dehydrogenase of Oligotropha carboxidovorans oxidises CO at SL041 a unique bimetallic [CuSMoO2] cluster in its active site (1). Post- "Four-dimensional" protein structures: examples from translational cluster assembly involves the gene functions metalloproteins coxDEFG. Insertional mutagenesis of coxD had the following Claudio Luchinat, CERM (Centre for Magnetic Resonance), consequences: (i) The resulting mutant OM5D::km was unable to University of Florence, Sesto Fiorentino (Florence), Italy. utilize CO under chemolithoautotrophic conditions, but (ii) retained Contact e-mail: [email protected] the ability to utilize H2. (iii) When grown with H2 and induced with CO, OM5D::km synthesized non-functional apo-CO The fact that an object, for example, a protein, possesses a three- dehydrogenase. (iv) EPR, EXAFS and crystallography revealed a dimensional structure seems an obvious concept. However, when [MoO3] centre in the active site of the apo-enzyme, which (v) could the object is flexible, the concept is less obvious. Proteins are be reconstituted to a functional [CuSMoO2] cluster employing indeed not rigid, but may sample more or less wide ranges of sulphide and Cu(I)-thiourea. different conformations. It has been recently proposed to call the CoxD is a MoxR-like AAA+ ATPase, enzymes involved in ATP range of sampled conformations the "fourth dimension" of a protein dependent processes of protein refolding and degradation (2) and a structure.1 A particular, but quite common, case of conformational member of the APE2220 phylogenetic subfamily of MoxR-proteins freedom is constituted by multidomain proteins, where two or more (3). The coxD gene is adjacent to coxE which contains a Von well structured and relatively rigid protein domains are tethered by Willebrand Factor Type A (VWA) domain, and both genes are part linkers that are more or less flexible. In most cases, the function of a cox gene cluster (4). These features separate the APE2220 itself depends on flexibility. Static structural information from, e.g., subfamily from the PA2707 subfamily of MoxR. We present X-ray, does not provide information on flexibility, and may even be evidence that CoxD operates as a molecular chaperone assisting misleading. Nuclear magnetic resonance is a better technique to CoxEFG and other essential functions to mediate the post- detect dynamics and assess conformational heterogeneity. We have translational insertion of sulphur and copper into the CO recently found that the presence of a paramagnetic metal in a dehydrogenase active site and the subsequent assembly to a metalloprotein permits the use of NMR in a novel way to describe catalytically competent [CuSMoO2] cluster. the conformational freedom of protein domains2 (i.e. their “four- [1] Dobbek et al. (2002) PNAS 99, 15971-6 ; [2] Iyer et al. (2004) dimensional” structure). A formally well defined way to define the J. Struct. Biol. 146, 11-31; [3] Snider J. and Houry WA. (2006) J. reciprocal arrangements of the domains in terms of their Maximum Struct. Biol. 156, 200-9; [4] Santiago et al. (1999) Gene 236, 115- Allowed Probability (MAP), is being proposed.3 24 1Marco Fragai M, Luchinat C, Parigi G, Acc Chem Res, 2006, 39:909-17. 2Bertini I, Calderone V, Cosenza M, Fragai M, Lee YM, Luchinat C, Mangani S, Terni B, Turano P, Proc Natl Acad Sci U S A, 2005 102:5334-9. 3Bertini I, Gupta Y, Luchinat C, Parigi G, Peana M, Sgheri L, Wang S, submitted

123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S59

SL044 SL046 Probing the mechanism of Formate Dehydrogenases Maturation of thiocyanate is promoted by its using EPR Mo(V) signals activator protein, P15K José J. G. Moura, REQUIMTE, Departamento de Química, Masafumi Odaka1, Takatoshi Arakawa1, Shota Hori1, Hiroyuki Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Mino2, Yoko Katayama3, Hiroshi Nakayama4, Naoshi Dohmae4, Caparica, Portugal. Masafumi Yohda1, 1Department of Biotechnology and Life Science, Contact e-mail: [email protected] Tokyo University of Agriculture and Technology, Tokyo, Japan; 2Division of Material Science (Physics), Graduate School of Formate dehydrogenase (Fdh) catalyzes the oxidation of formate to Science, Nagoya University, Nagoya, Japan; 3Department of carbon dioxide. The crystal structures of three Fdhs have been Environment and Natural Resource Science, Tokyo University of reported: E.coli Mo-Fdh-H and E.coli Mo-Fdh-N and D.gigas W- Agriculture and Technology, Tokyo, Japan; 4Biomolecular Fdh. The catalytic subunit contains the active site and one [4Fe-4S] characterization team, RIKEN, Saitama, Japan. cluster. The oxidized active site is coordinated to 2xMGD, one Se Contact e-mail: [email protected] atom from a selenocysteine, and a sixth ligand X, identified as a hydroxyl in Ec Fdh-N, Ec Fdh-H, and D. desulfuricans Fdh, but the Nitrile hydratase (NHase) family proteins are Co- or Fe-containing Dg Fdh X-ray data suggest the presence of a S atom. A reevaluation enzymes having two post-translationally modified cysteine ligands, of the crystallographic data of the Ec Fdh-H reduced by formate in cysteine-sulfinic acid (Cys-SO2H) and cysteine-sulfenic acid (Cys- the presence of azide proposed that this ligand is better fitted as a SOH). NHase family proteins require their specific activator sulfur than a oxygenic ligand (Raaijmakers and Romão, JBIC proteins for the functional expression. Here, we show the function (2006)). We report detailed EPR studies on as-prepared, formate of the activator protein, P15K, for thiocyanate hydrolase (SCNase), reduced and azide inhibited samples of Dd Fdh, in order to compare a Co-type NHase family protein. When P15K was co-expressed with reduced Ec Fdh-H in the presence of azide. The re-evaluation with Į, ȕ or Ȗ subunit of SCNase in Escherichia coli, only the Ȗ of the 3D structure of the formate reduced Ec Fdh-H in the presence subunit which contained all ligand residues formed a stable of azide shows that the SeCys ligand is not coordinated to Mo. complex (ȖP15K) with a stoichiometry of Ȗ: P15K = 1: 1. It is of Based on this observation it was suggested a completely different interest that the Ȗ subunit was expressed as inclusion bodies when it mechanism. In the presence of formate and absence of inhibitors the was expressed solely. Then, we studied the effect of Co ion on the EPR signals assigned to competent catalytic species are different expression of ȖP15K. When ȖP15K was expressed in the Co- from those obtained in presence of azide, we will focus on the enriched medium, the isolated Ȗ subunit as well as ȖP15K was clarification of mechanistic aspects of Fdhs. Relevant references obtained. Both the isolated Ȗ subunit and ȖP15K incorporated will be given at the presentation. significant amount of Co ions and possessed the Cys-SO2H Acknowledgments: MG Rivas, P González, C Brondino, I Moura, modification like native SCNase. Considering that P15K has MJ Romão. FCT-MCTES, SEPCYT, CAI+D-UNL. significant amino acid sequence similarity with the ȕ subunit and that the metal-binding site is located at the interface between ȕ and Ȗ subunits, the function of P15K is to associate with the Ȗ subunit SL045 and to assist the Co-incorporation as well as the generation of the Impact of heme to protein linkages in peroxidases on cysteine modifications. We have been trying to reconstitute SCNase redox chemistry and catalysis from ȖP15K and the Ȗ subunit. Based on the results obtained, we Christian Obinger1, Jutta Vlasits1, Marcia Bellei2, Gianantonio will discuss the maturation mechanism of the Co-type NHase Battistuzzi2, Paul G. Furtmüller1, 1BOKU - University of Natural family proteins. Resources and Applied Life Sciences, Department of Chemistry, Vienna, Austria; 2University of Modena and Reggio Emilia, Department of Chemistry, Modena, Italy. SL047 Contact e-mail: [email protected] Towards a protocol for solution structure determination of copper(II) proteins through protonless 13C direct- The mammalian peroxidases myeloperoxidase (MPO), eosinophil detection NMR peroxidase, lactoperoxidase (LPO) and thyroid peroxidase Roberta Pierattelli, University of Florence, Dept of Chemistry and participate in host defence against infection, hormone synthesis and CERM, Sesto Fiorentino, Italy. pathogenesis. The most striking feature of these heme enzymes is Contact e-mail: [email protected] the existence of two covalent ester bonds between the prosthetic group and the protein in the functional, mature proteins. Nuclear magnetic resonance spectroscopy plays a prominent role in Myeloperoxidase is unique in having an additional vinyl-sulfonium the characterization of proteins and their interactions with other bond. Based on the crystal structures of human MPO and its molecules. Solution structures have been determined for several complexes with cyanide, bromide and thiocyanide as well as on the paramagnetic systems by using 1H NMR-based experiments, and in structure of bovine LPO, the impact of heme distortion and several cases paramagnetic effects have been exploited to derive asymmetry on the spectral and enzymatic properties is discussed as structural information useful for refinement of a structure.1 is the role of the MPO-typical electron withdrawing sulfonium ion The magnitude of paramagnetic effects on nuclear relaxation rates linkage in raising the reduction potential of its redox intermediates depends on the nature of the metal ion and its electronic relaxation and maintaining a rigid solvent network at the distal heme cavity. rate. Depending on the metal-nucleus distance, electron-nucleus These structural features allow MPO to be the singular human coupling effects may prevent the observation of the proton signals enzyme that efficiently binds and oxidizes chloride to antimicrobial of residues close to the metal site, due to induced broadening of the hypochlorous acid. NMR lines. This is the case for type II copper(II)-containing Arnhold, J., Monzani, E., Furtmüller, P. G., Zederbauer, M., proteins, where protons close to the paramagnetic copper center are Casella, L., Obinger, C. Kinetics and thermodynamics of halide and characterized by relaxation rates too large to be studied with a nitrite oxidation by heme peroxidases. Eur. J. Inorg. Chem. 19 conventional NMR approach.2 (2006), 3801-3811 Carbon-13 direct-detection NMR offers advantages for the study of paramagnetic proteins because paramagnetic dipolar contributions to nuclear relaxation depend on the square of the gyromagnetic ratio of the observed nucleus; thus, a switch from 1H to 13C detection guarantees a sizable decrease in relaxation rates.3;4 This is demonstrated in Cu,Zn , where 13C direct- detection NMR experiments enable detection of resonances as close 123 S60 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

as 6 Å from the metal ion, and allow determination of structural disulfide connectivities (5-13, 17-29 and 22-27) and the three restraints necessary for structure refinement.5 dimensional structure were elucidated by high field NMR [Skjeldal Solid-state NMR, that is developed on 13C direct-detection, provides et al (2002)Arch.Biochem.Biophys., 399, 142-148]. The peptide an additional tool for the NMR investigation of Cu(II)-containing binds to the micelle surface with relatively high affinity [Shenkarev proteins.6 et al (2006) FEBS J.273, 2658-2672]. Rerefences We have also found that the peptide is able to bind manganese in a 1) Bertini, I.; Luchinat, C.; Parigi, G.; Pierattelli, R. ChemBioChem specific metal binding site, which could be a binding site for 2005, 6, 1536-1549. calcium. The structure is deposited in PDB as 1JJZ. 2) Banci, L.; Pierattelli, R.; Vila, A. J. Adv.Prot.Chem. 2002, 60, 397-449. 3) Bermel, W.; Bertini, I.; Felli, I. C.; Kümmerle, R.; Pierattelli, R. SL050 J.Am.Chem.Soc. 2003, 125, 16423-16429. Redox properties of the c-type heme centers in the novel 4) Bermel, W.; Bertini, I.; Felli, I. C.; Piccioli, M.; Pierattelli, R. PQQ-dependent alcohol dehydrogenase from nitrogen Progr.NMR Spectrosc. 2006, 48, 25-45. fixing Gluconacetobacter diazotrophicus 5) Bertini, I., Felli, I. C., Luchinat, C., Parigi, G.; Pierattelli, R. 1 2 Martha E. Sosa-Torres , Saúl Gómez-Manzo , Edgardo submitted. 2 3 1 Escamilla-Marván , Peter M. H. Kroneck , Facultad de Química, 6) Pintacuda, G.; Giraud, N.; Pierattelli, R.; Böckmann, A.; Bertini, Universidad Nacional Autónoma de México, México D.F., Mexico; I.; Emsley, L. Angew.Chem.Int.Ed. 2007, 46, 1079-1082 2 Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México D.F., Mexico; 3Fachbereich Biologie, Universität SL048 Konstanz, Konstanz, Germany. Contact e-mail: [email protected] The axial ligand and associated hydrogen bonding network in the distal site of control the Multi-heme proteins and enzymes are important constituents of dynamical freedom of the enzyme during catalysis bacterial electron transfer chains and are involved in many Mario Rivera1, Juan C. Rodriguez2, 1University of Kansas, R. N. respiratory processes. In sulfate-reducing bacteria the tetraheme Adams Institute for Bioanalytical Chemistry and Departemt of protein cytochrome c3 and the nonaheme cytchrome c play Chemistry, Lawrence, KS, USA; 2University of Kansas, R. N. important roles in transferring electrons between the [Ni,Fe]- Adams Institute for Bioanalytical Chemistry and Department of hydrogenase and membrane-bound respiratory complexes [1]. In Chemistry, Lawrence, Kansas, KS, USA. nitrogen-fixing bacteria, such as Gluconacetobacter diazotrophicus, Contact e-mail: [email protected] a multi-heme PQQ-dependent alcohol dehydrogenase (PQQ-ADH) may also serve as a protectant for the oxygen-labile nitrogenase [2]. NMR experiments have been used to investigate ȝs-ms motions in The PQQ-ADH of G. diazotrophicus is a heterodimer composed of heme oxygenase from Pseudomonas aeruginosa (pa-HO) in its SU I (PQQ cofactor and one c-type cytochrome) and SU II (three c- ferric state, inhibited by CN¯ (pa-HO-CN) and N3¯ (pa-HO-N3), type cytochromes). This latter PQQ-ADH is rather unique in the and in its ferrous state, inhibited by CO (pa-HO-CO). Comparative sense that the heme centers remain in the reduced Fe(II) state even analysis of the data indicates that the distal ligand affects the when isolated in the presence of dioxygen. We will describe the conformational freedom of the polypeptide in regions removed from redox properties of the four different c-type cytochromes in the the heme. Interpretation of the dynamical information in the context ADH from G. diazotrophicus by applying UV/Vis- and EPR of the structure of pa-HO shows that secondary structure harboring spectroscopy-monitored redox titrations. Preliminary residues involved in the network of structural H-bonded waters electrochemistry measurements are also included. An undergo ȝs-ms motions in pa-HO-CN, which was studied as a intramolecular electron transfer (IET) pathway between the c-type model of the resting state form. In comparison, similar motions are heme centers within subunits I and II, and to the ubiquinone is suppressed in the pa-HO-CO and pa-HO-N3 complexes, which proposed. were studied as mimics of the oxyferrous and ferric hydroperoxide [1] Matias P.M., Pereira I.A.C., Soares C. M., Carrondo M. A. intermediates. These findings suggest that in addition to proton (2005) Prog. Biophys. Mol. Biol. 89: 292. delivery to the nascent FeIII-OO¯, the H-bonding network serves [2] Flores-Encarnacion M., Contreras-Zentella M., Soto-Urzua L., two additional roles: (i) propagate the electronic state in each of the Aguilar G.R., Baca B.E., Escamilla J.E. (1999) J. Bacteriol. catalytic cycle steps to key but remote sections of the polypeptide 181:6987.; González B., Martínez S., Chávez J.L., Lee S., Castro via small rearrangements and (ii) modulate the conformational N.A., Domínguez M.A., Manzo S., Contreras M.L., Kennedy C., freedom of the enzyme, thus allowing it to adapt to the demanding Escamilla J.E. (2006) Biochim. Biophys. Acta - Bioenergetics, 1757: changes in axial coordination and substrate transformations taking 1614. place during a catalytic cycle. This idea was probed by disrupting the H-bonding network by replacing R80 for L. rc-CPMG studies revealed that the mutant exhibits nearly global conformational SL051 motions that are absent in wild type pa-HO-N3. These “chaotic” Histidine-rich protein, Hpn and metallothionein: are they motions in R80L-pa-HO-N3 are likely related to its significantly similar? lower efficiency to hydroxylate heme. Hongzhe Sun1, Ruiguang Ge1, Yibo Zeng1, Hui Wang1, Jiandong Huang2, 1Department of Chemistry, The University of Hong Kong, Hong Kong, China; 2Department of Biochemistry, The University SL049 of Hong Kong, Hong Kong, China. Metal binding site in the macrocyclic peptide Kalata Contact e-mail: [email protected] Lars Skjeldal, Norwegian University of Life Sciences (UMB), Dept. of Chemistry, Biotechnology and Food Science, Aas, Helicobacter pylori (H. pylori), a Gram-negative, and spiral-shaped Norway. bacterium, has been known to be the causative agent of chronic Contact e-mail: [email protected] gastritis and peptic ulcers. The organism produces a large amount of a nickel-containing enzyme urease, which is believed to neutralize Metal binding site in the macrocyclic peptide Kalata. gastric acid by producing ammonia for the survival of the The African medical plant Oldenlandia affinis, was used as a bacteria.[1,2] A constant supply of nickel is therefore essential for the decoction by the natives during child-birth to support labour. The synthesis and activity of nickel-containing enzymes. Several delivery time was remarkable short, and the uterine contractions histidine-rich proteins and domains have been found in H. pylori.[2] extremely strong. The active compound in the plant is the cyclic Hpn (28 His-residues out of 60 aa) is a small cytoplasmic protein peptide Kalata B1, which contains three disulfide linkages. The and is present as a multimer with 20-mer being the predominant 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S61

species in solution and binds to five Ni2+ and four Bi3+ per ORAL PAPERS [3] monomer moderately (Kd of 7.1 and 11.1 ȝM respectively). Although in vitro, it binds to Cu2+ stronger than Ni2+ and Bi3+, the in O025 2+ 3+ 2+ [4] vivo protection by the protein is in the order of Ni > Bi > Cu . Resonance Raman Spectroscopic Studies on the Alkaline Hpn may therefore serve to buffer intracellular Ni2+ in much the same way as that the small and cysteine-rich protein, Transition of a Blue Copper Protein, Pseudoazurin and metallothionein interacts with Zn2+/Cu+.[5,6] Its M16X Variants Rehab F. Abdelhamid1, Takamitsu Khozuma2, 1Institute of Acknowledgement: This work was supported by RGC, AoE of 2 UGC and the University of Hong Kong! Applied Beam Science, Ibaraki University, Mito, Japan; Institute [1] Covacci A et al. (1999) Science 284, 1328-1333. of Applied Beam Science, Ibaraki University, , 2-1-1 Bunkyo, Mito, [2] Tomb J-F et al. (1997) Nature 388, 539-547. Ibaraki 310-8512,, Japan. [3] Ge R et al. (2006) Biochem. J. 393: 285-293. Contact e-mail: [email protected] [4] Ge R et al. (2006) J. Am. Chem. Soc. 128: 11330. [5] Vašák MJ, Hasler DW (2000) Curr. Opin. Chem. Biol. 4: 177- Several blue copper proteins involving pseudoazurin (PAz) shows a 183 structural transition at alkaline pH. The spectroscopic and [6] Wang H et al. (2006) FEBS Lett. 580: 795-800 electrochemical studies of the alkaline transition of PAz were reported [1, 2]. Very recently the effect of the second sphere interaction in the vicinity of the active site of PAz M16X variants, SL052 M16Y, M16F, M16W, and M16V has been reported [3]. Resonance Raman (RR) spectroscopic studies of wild-type and the M16X The Structure of the Hyponitrite Species in the Heme Fe- variants were performed to know the details of the effect of second CuB Center of ba3 Oxidase from Thermus thermophilus sphere interaction on the alkaline transition of pseudoazurin. RR Costas Varotsis1, Takehiro Ohta2,3, Teizo Kitagawa2, Tewfik 4 5 1 spectra of wild-type and M16X mutants at pH 7.2 and 10.2 were Soulimane , Eftychia Pinakoulaki , University of Crete, -1 2 measured in the frequency region of 200-550 cm . The RR spectra Department of Chemistry, Heraklion, Greece; Okazaki Institute for of the wild-type and the M16X spectra at pH 7.2 showed five Integrative Bioscience, National Institutes of Natural Sciences, -1 3 intense Raman bands at 418, 398, 381, 367, and 355 cm and Okazaki, Japan; Stanford University, Department of Chemistry, shoulder bands at 335, 439, and 454 cm-1 except M16Val. The RR Stanford, CA, USA; 4University of Limerick, College of Science, 5 spectra of wild-type and M16X variants indicated the disappearance Limerick, Ireland; University of Cyprus, Department of Chemistry, of several Raman bands at pH10.2. Nicosia, Cyprus. This work was supported by the Project of Development of Basic Contact e-mail: [email protected] Technologies for Advanced Production Methods Using Microorganism Functions by the New Energy and Industrial The formation of the hyponitrite species from two NO molecules is Technology Development Organization (NEDO). the key intermediate in the reduction of NO to N2O in References denitrification. The reaction is used by bacteria as an alternative to [1] Takamitsu Kohzuma et al. J. Biol. Chem. 270, 1995. oxygen-based respiration. The bacterial reductase (Nor) [2] Katsuko Sato et al. Biochemistry. 41, 2002. and the ba3- from Thermus thermophilus catalyze the [3] Rehab Abdelhamid et al. JBIC. 12, 2007. reduction of NO to N2O. Here we report on the formation of the hyponitrite species in the heme a3 Fe/CuB binuclear center of ba3- oxidoreductase from T. thermophilus. The N-N bond (ȞN-N= 1334 O026 cm-1) of the hyponitrite-bound to the heme Fe is detected only under 244 nm Raman excitation, and the bending vibration of heme Fe-N- Crystallographic and spectroscopical studies of peroxide- -1 derived myoglobin intermediates OH at 624 cm under 428 nm Raman excitation. The ȞN-N is 1 2 1 sensitive to pH and pD exchanges indicating the interaction of the Hans Petter Hersleth , Åsmund K. Røhr , Carl Henrik Görbitz , Karl Kristoffer Andersson2, 1University of Oslo, Chemistry, Oslo, hyponitrite species with a H2O molecule. We propose a mechanism 2 for the formation of the hyponitrite species that is initiated by the Norway; University of Oslo, IMBV, Oslo, Norway. Contact e-mail: [email protected] reduction of CuB by NO. With the identification of the hyponitrite species the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification is fully supported and the High resolution crystal structure of myoglobin in the pH range 5.2- - + 8.7 [1-4] have been used as models for the peroxide-derived mechanism for the 2e /2H reduction of NO to N2O can be described with more certainty. compound II intermediates in haem peroxidases and . References The observed Fe-O bond length (1.86-1.90Å) is consistent with a [1] Pinakoulaki, E., Ohta, T., Soulimane, T., Kitagawa, T. & single bond. This finding is supported by observation of a new low energy 18O-peroxide-sensitive resonance Raman Fe-O mode at 687 Varotsis, C. J. Am. Chem. Soc. 127, 15161-15167 (2005). -1 [2] Ohta, T., Kitagawa, T. & Varotsis, C. Inorg. Chem. 45, 3187- cm [4]. We observe some radiation-induced changes in both 3190 (2006). compound II (forming a novel semi-stable intermediate H) and in [3] Varotsis, C., Ohta, T., Kitagawa, T., Soulimane, T., & the resting ferric state of myoglobin by single-crystal Pinakoulaki, E. Angew. Chem. Int. Ed. 46, 2210-2214 (2007). microspectrophotometry [4]. Our data suggest for myoglobin the compound II intermediate consists of a FeIV-O species with a single bond. The presence of FeIV is indicated by Mössbauer spectroscopy [4], and quantum refinements and DFT-calculations suggest that this species is protonated [2,3]. The presence of a FeIV-OH state seems to be general for compound II intermediates. Additionally, an isoelectronic form of the compound 0 intermediate has been generated by radiation-induced changes (to be submitted J. Am. Chem. Soc). [1] Hersleth, H.-P., Dalhus, B., Görbitz, C. H., and Andersson, K.K.(2002) J. Biol. Inorg. Chem. 7, 299-304 [2] Nilsson, K., Hersleth, H.-P., Rod, T. H., Andersson, K. K., and Ryde, U. (2004) Biophys. J. 87, 3437-3447 [3] Hersleth, H.-P., Ryde, U., Rydberg, P., Görbitz, C. H., and Andersson, K. K. (2006) J. Inorg. Biochem. 100, 460-476 123 S62 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

[4] Hersleth, H.-P., Uchida, T., Røhr, Å. K., Teschner, T., O029 Schünemann, V., Kitagawa, T., Trautwein, A. X., Görbitz, C. H., Biogenesis of iron-sulfur clusters and intracellular iron J. Biol. Chem and Andersson, K. K. (2007) . Submitted metabolism under oxidative stress conditions Huangen Ding, Juanjuan Yang, Liana C. Coleman, Louisiana State University, Baton Rouge, LA, USA. O027 Contact e-mail: [email protected] Redox thermodynamics of the Fe3+/Fe2+ couple in wild- type and mutated heme peroxidases Iron is an essential element for living organisms. Ironically, iron is Gianantonio Battistuzzi1, Marzia Bellei1, Christa Jakopitsch2, Jutta highly toxic to cells since free iron can promote the production of Vlasits2, Paul G. Furtmüller2, Marco Sola1, Christian Obinger2, hydroxyl free radicals via the Fenton Reaction. Here we reported 1University of Modena and Reggio Emilia, Modena, Italy; 2BOKU the interplay of the iron binding between the iron-sulfur cluster – University of Natural Resources and Applied Life Sciences, assembly protein IscA and the Friedreich's ataxia protein frataxin, Wien, Austria. under physiological and oxidative stress conditions. Previous Contact e-mail: [email protected] studies indicated that both IscA and frataxin are able to bind iron. However, their iron binding mechanisms are very different. In IscA, The thermodynamics of the one-electron reduction of the ferric the invariant three cysteine resideus are required for the iron heme in wild-type and mutated heme Synechocystis - binding. On the other hand, the carboxyl groups of the highly peroxidase and human myeloperoxidase were determined through conserved aspartate and glutamate residues in frataxin are essential spectro-electrochemical experiments [1, 2]. The data are interpreted for the iron binding. In this study, we have shown that in the in terms of ligand binding features, electrostatic effects and presence of the thioredoxin reductase system which emulates solvation properties of the heme environment. Reduction-induced intracellular redox potential, frataxin fails to bind any iron even at a solvent reorganization effects turn out to be the major effectors of 10-fold excess of iron. IscA, on the other hand, can efficiently reduction entropy and influence reduction enthalpy, which mainly recruit iron and transfers the iron for the iron-sulfur cluster depends on the anionic character of the proximal histidine and the assembly in IscU. In the presence of hydrogen peroxide, however, polarity of the heme environment. IscA completely loses its iron binding activity due to the oxidation [1] M. Bellei, C. Jakopitsch, G. Battistuzzi, M. Sola, C. Obinger of the thiol groups, whereas frataxin becomes a competent iron- (2006) Biochemistry 45, 4768-4774. binding protein and attenuates the iron-mediated production of [2] G. Battistuzzi, M. Bellei, M. Zederbauer, P. G. Furtmüller, M. hydroxyl free radicals. The results suggest that IscA is capable of Sola, C. Obinger (2006) Biochemistry, 45, 12750-12755. recruiting intracellular iron for the iron-sulfur cluster assembly under normal physiological conditions, whereas frataxin may serve as an iron chaperon to sequester redox active free iron and alleviate O028 cellular oxidative damage under oxidative stress conditions. Structural & Biological Analysis of the Metal Sites from the Escherichia coli Hydrogenase Accessory Protein O030 HypB Alistair V. Dias, Deborah B. Zamble, University of Toronto, Assignment of individual metal redox states in Toronto, ON, Canada. metalloproteins by crystallographic refinement at Contact e-mail: [email protected] multiple X-ray wavelengths Oliver Einsle1, Susana L. A. Andrade1, Holger Dobbek2, Jacques The [NiFe]-hydrogenase protein expressed in many bacteria such as Meyer3, Douglas C. Rees4, 1Institute for Microbiology and E. coli and H. pylori is a protein that contains an intricate metal core Genetics, Göttingen, Germany; 2Laboratorium required for the reversible production of two protons and two Proteinkristallographie, Bayreuth, Germany; 3CEA-Grenoble, electrons from hydrogen gas. It is known that the assembly of the Grenoble, France; 4California Institute of Technology, Pasadena, hydrogenase metal cluster involves the coordinated activity of a CA, USA. number of helper proteins to deliver all of the correct components Contact e-mail: [email protected] and assemble the metallocenter correctly (1). However, very little is known about the mechanisms of action of the specific components, In recent years X-ray crystallography revealed the existence and how they interrelate or interact with each other, or how metal fine structure of numerous multi-metal and multi-center metal selectivity is achieved. clusters in proteins. X-ray data are routinely collected at or around The accessory protein, HypB, is necessary for Ni(II) insertion in to absorption edge in order to exploit anomalous scattering for phase the NiFe cluster of hydrogenase in E. coli. By using XAS data and determination, but the additional information contained in these mutagenic studies we have been able to form a structural picture of edges - as used in X-ray absorption spectroscopy - is commonly not the high and low-affinity metal sites present in the HypB protein utilized in crystallography. (2). We have determined the amino acid ligands in addition to the We have established a method for refining anomalous scattering geometry around the Ni(II) ion in both the high-affinity and low- contributions for individual atoms within a crystal structure for affinity metal site. The low-affinity site, a possible structural site, multiple data sets collected along an absorption edge [1], yielding has a stronger preference for Zn(II) and the ligands and geometry information on redox state and electron distribution at each atom. for this metal were also resolved. Further analysis also indicates that This allows for the detailed analysis of redox events and the tracing there may be communication between the two metal domains. of electron flow within protein systems. The test case of an These data provides valuable insight into the structural antiferromagnetically coupled [2Fe:2S] cluster is presented, where a configuration of HypB and the mechanism of Ni(II) delivery into mixed-valence reduced state with localized charges confirms the hydrogenase. reliability of the approach, and implications for highly complex 1. S. Lutz et. al. (1991) Mol. Microbiol. 5,123-35. metal clusters are discussed. 2. M. R. Leach et. al. (2005) Biochemistry, 44, 12229-38. [1] Einsle, O., Andrade, S.L.A., Dobbek, H., Meyer, J. & Rees, D.C. (2007) J. Am. Chem. Soc. 129, 2210-2211.

123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S63

O031 O033 Investigation of the coordination structures of the Unnatural Amino Acid Containing Mutants of KatG molybdenum(V) sites ofsulfite oxidizing enzymes by Exhibit Enhanced Peroxidase Activity pulsed EPR spectroscopy: Direct detection of coordinated Rania Dumarieh, Reza A. Ghiladi, North Carolina State University, sulfate using 33S labeling Raleigh, NC, USA. John H. Enemark1, Andrei V. Astashkin1, Arnold M. Raitsimring1, Contact e-mail: [email protected] Kayunta Johnson-Winters1, Eric L. Klein1, Robert S. Byrne2, Russ Hille2, Heather L. Wilson3, K. V. Rajagopalan3, 1University of Catalase-peroxidases (KatG) are bifunctional hemoproteins Arizona, Tucson, AZ, USA; 2The Ohio State University, Columbus, possessing both catalase and peroxidase activities. The crystal OH, USA; 3Duke University Medical Center, Durham, NC, USA. structures of different KatGs each revealed the presence of a novel Contact e-mail: [email protected] active site modification comprised of two covalent bonds between three amino acid side chains: Trp107, Tyr229, and Met255 Sulfite oxidizing enzymes (SOEs) are physiologically vital and (Mycobacterium tuberculosis numbering). Absent from the occur in all forms of life. During the catalytic cycle the five- peroxidases, this Met-Tyr-Trp ‘crosslink’ has been suggested to coordinate square-pyramidal oxo-molybdenum active site passes impart catalase activity to the KatGs, although how it carries out through the Mo(V) state, and intimate details of the structure can be this function has been the subject of recent debate. obtained from pulsed EPR spectroscopy through the hyperfine To better understand the role which the crosslink plays in enzyme interactions (hfi) and nuclear quadrupole interactions (nqi) of catalysis, we have expressed several KatG(Tyr229UAA) mutants, nearby magnetic nuclei (e.g., 1H, 2H, 17O, 31P, 33S). By employing where UAA represents the following unnatural amino acids: p-Ac, spectrometer operational frequencies ranging from ~4 to ~32 GHz, p-NH2, p-N3, p-Br, p-I, and p-OMe derivatives of phenylalanine. it is possible to make the nuclear Zeeman interaction significantly Notably, these mutants exhibit up to a 300-fold increase in greater than the hfi and nqi, and thereby simplify the interpretations peroxidase catalytic efficiency (kcat/Km) compared to the WT of the spectra. The SOEs exhibit three general types of Mo(V) enzyme (see Table). Specifically, kcat was enhanced by ~20-60 fold, structures which differ in the number of nearby exchangeable whereas Km was improved slightly (~1-8 fold), indicating that the protons (one, two or zero). The CW-EPR and electron spin echo gain in catalytic efficiency is mainly from increasing the rate of the envelope modulation (ESEEM) data for sulfite oxidase from peroxidase reaction, with only a minor contribution from improving Arabidopsis thaliana (At-SO) that has been reduced to Mo(V) at pH substrate binding. While these KatG mutants were unable to form = 6 with sulfite labeled with 33S (I = 3/2) show no exchangeable Met-UAA-Trp crosslinks, resulting in loss of catalase activity, they 33 nevertheless represent one of the first examples in hemoprotein protons and a S hyperfine interaction whose magnitude (aiso = 4.8 MHz) demonstrates the presence of coordinated sulfate, a engineering where an enzymatic activity has been enhanced through postulated intermediate in the catalytic cycle of sulfite oxidizing the use of UAAs. Details of these and our complementary stopped- enzymes. The fatal R160Q mutant of human sulfite oxidase also flow UV-visible spectroscopic studies will be presented. shows 33S couplings from bound sulfate. These are the first Peroxidase Activity of WT KatG and Selected Mutants -1 -1 -1 examples of direct detection of coordinated sulfate in molybdenum kcat, s Km, mM kcat/Km, (M s ) Normalized 33 enzymes and only the second case of pulsed EPR detection of a S WT 0.062 ± 0.001 8.44 ± 0.45 7.3 ± 0.4 1 - metal ion interaction in metalloenzymes. p-Br-F 1.27 ± 0.08 1.50 ± 0.21 849 ± 99 116 p-Ac-F 2.37 ± 0.01 2.60 ± 0.04 910 ± 12 124 O032 p-I-F 1.70 ± 0.02 1.57 ± 0.08 1083 ± 55 148

Role of highly conserved three-histidines ligand p-NH2-F 3.46 ± 0.23 2.37 ± 0.23 1461 ± 141 200 environment of type-2 copper site in copper containing p-OMe-F 2.17 ± 0.07 1.13 ± 0.07 1920 ± 120 263

nitrite reductase p-N3-F 2.36 ± 0.03 1.09 ± 0.06 2155 ± 117 295 Hiroshi Fujii, Masato Kujime, National Institutes of Natural Sciences, Okazaki, Japan. Contact e-mail: [email protected] O034 Denitrification is an important component of the global nitrogen Design, synthesis, and structures of tethered substrates as cycle that is responsible for regulating the amount of fixed nitrogen probes of the active sites of cytochrome P450cam, available for plant growth. In denitrification process, the reduction cytochrome c peroxidase, and . of NO2- to NO is catalyzed by nitrite reductase, containing either David B. Goodin1, Edith Glazer1, Richard Wilson1, Andrew heme cd1 or copper. The x-ray crystal structures of copper- Annalora1, Stefan Vetter2, Harry B. Gray3, 1The Scripps Research containing nitrite reductases (Cu-NiRs) show a common reaction Institute, La Jolla, CA, USA; 2Florida Atlantic University, Boca center, containing a pair of copper ions, type-1 Cu and type-2 Cu. Raton, FL, USA; 3California Institute of Technology, Pasadena, The type-1 Cu site has (His)2-Cys-Met donor set, which is typical CA, USA. for electron-transfer copper proteins. On the other hand, the type-2 Contact e-mail: [email protected] Cu site has three histidine ligands, (His)3, and a water as the fourth ligand in the resting state. The type-2 Cu site has been suggested to We are exploring the use of synthetic molecular wires to probe the be a reaction center where nitrite is reduced to NO with an electron active site and function of heme enzymes including P450cam, transferred from the type-1 Cu site. The (His)3 coordination cytochrome c peroxidase (CcP), and nitric oxide synthase (NOS). environment is highly conserved in all Cu-NiRs. In order to These wires, consisting of ligand analogs tethered to a reporter or investigate the functional role of the (His)3 coordination substrate, are designed to bind specifically to the active site channel environment in the type-2 Cu site, we set out to prepare copper(I)- of a given enzyme. Adamantane analogs linked through nitrite complexes from sterically hindered tris(4-imidazolyl)carbinol hydrocarbon or polyether linkers bind in the active site channel of (TIC) and tris(pyrazolyl)methane (TPM) ligands to mimic the P450cam, inducing a large range of conformational changes in the F (His)3 coordination environment in the type-2 Cu site of CuNiR. and G helix regions. Benzimidazole analogs attached to peptide Particularly, the copper(I) nitrite complex having sterically hindered linkers bind to an engineered channel in place of the electron tris(4-imidazolyl)carbinol is expected to be the best model complex transfer pathway of CcP, allowing the electron transfer to be probed for the copper(I) nitrite complex in Cu-NiR. We report here though an electrode interface. Finally, analogs of 6(R)-tetrahydro- synthesis, structural features, spectroscopic properties, and L-biopterin (H4B) linked to Ru based photosensitizers bind to reactivity of copper(I) nitrite complexes with sterically hindered pterin free iNOS. We will report on the synthesis, structures, TIC and TPM ligands. binding and functional properties of these compounds to their target enzymes. 123 S64 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

O035 aldehyde oxidizing activities [2]. One of the enzymes, Redox properties of the Fe-hydrogenase homologue Nar1 formaldehyde : ferredoxin oxidoreductase (FOR) activates free (i.e. from Saccharomyces cerevisiae non-hydrated) formaldehyde [3]. In addition to a tungsto-bispterin Peter L. Hagedoorn1, G. P. H. van Heusden2, H. Y. Steensma2, W. cofactor these enzymes contain an electron transferring iron-sulfur R. Hagen1, 1Department of Biotechnology, Delft University of cubane that heavily dominates the optical spectra making Technology, Delft, The Netherlands; 2Institute of Biology Leiden, mechanistic studies through optical monitoring difficult. EPR Leiden University, Leiden, The Netherlands. monitoring of paramagnetic W(V) intermediates is not a viable Contact e-mail: [email protected] alternative since substrate oxidation is essentially an electron pair reaction coupled to the one-step reduction of W(VI) to W(IV) [3]. However, molybdopterin and tungstopterin cofactors do exhibit Fe-hydrogenases are hydrogen producing and consuming enzymes -1 -1 that occur in bacteria and anaerobic unicellular eukaryotes. The relatively weak optical absorptions, typically İvisible § 2 cm mM , catalytic site is a unique iron-sulfur cluster with organometallic CO and this property has been used previously in a pre steady state and CN ligands that has been named the H-cluster [1]. Genomic kinetics study on DMSO reductase, an enzyme that has a single information from higher eukaryotes revealed a family of proteins molybdopterin cofactor only. We have now identified substrate- P. furiosus (Narf-family) that share significant homology with the Fe- induced weak optical changes of the tungstopterin in hydrogenases. All the amino acids that are involved in the FOR, and we have used these for monitoring pre-steady state coordination of the H-cluster are conserved in the Narf-family. kinetics employing stopped-flow absorption spectrometry. The Recently convincing evidence has emerged of involvement of the results, combined with tryptophan stopped-flow fluorescence and S.cerevisiae Narf-family member, Nar1, in cytosolic iron-sulfur with data from steady-state kinetics and from EPR titrations, form cluster biosynthesis [2]. In order to study the iron-sulfur clusters of the basis for a proposal for the catalytic cycle of this W/Fe/S Nar1 we have cloned and over-expressed the protein in S.cerevisiae enzyme. and E.coli. EPR monitored redox titrations were performed to [1] Bevers et al. (2006) J. Bacteriol. 188: 6498. characterize the iron-sulfur cluster(s) and additional paramagnetic [2] Bevers et al. (2005) J. Bacteriol. 187: 7056. centers in the protein. Evidence of substoichiometric [3Fe-4S] [3] Bol et al. (2006) J. Biol. Inorg. Chem. 11: 999. cluster and stoichiometric mononuclear Fe and [4Fe-4S] cluster was found (Fig. 1). Furthermore evidence for oxidative FeS cluster loss and subsequent protein aggregation was found. O037 [1] D.S. Horner, B. Heil, T. Happe, T.M. Embly, Trends Biochem. UV resonance Raman investigation of the functional Sci. 2002, 27, 148-153. mechanism of Ec DOS protein: Utilization of the heme 6- [2] J. Balk, A.J. Pierik, D.J. Netz, U. Muhlenhoff, R. Lill., EMBO propionate hydrogen bond network J., 2004, 23, 2105-2115. Teizo Kitagawa1, Samir F. El-Mashtoly1, Hiroto Takahashi2, Toru Shimizu2, 1Okazaki Institute for Integrative Bioscience, Okazaki, Japan; 2Institue of Multidisciplinary Research Advanced Material, Tohoku University, Sendai, Japan. Contact e-mail: [email protected]

The direct oxygen sensor protein from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of O2 or CO to a heme enhances the PDE activity towards c-di-GMP. We report ultraviolet resonance Raman (UVRR) spectroscopic investigations of the reduced, O2- and CO-bound forms of its heme-bound PAS domain (Ec DOSH). The UVRR results show that heme discriminates different ligands, resulting in altered conformations in the protein moiety. Specifically, the environment around Trp53 that contacts heme 2-vinyl group, shifts towards more hydrophobic upon O2 binding, while towards more hydrophilic upon the CO-binding. The PDE activity of the O2- and CO-bound forms for W53F mutant is significantly decreased compared with that of WT, demonstrating the importance of Trp53 in its function. Binding of O2 or CO to the heme produces drastic changes in the Tyr126 at the surface of the sensor domain. Furthermore, we found that Asn84 forms a hydrogen bond with Tyr126 either in the O2- or CO-bound forms but not in the reduced form. The PDE activities of the ligand bound forms for Asn84Val and Tyr126Phe mutants are significantly reduced compared with that of WT. suggesting the importance of the hydrogen-bonding network from heme 6-propionate to Tyr 126 through Asn84 in signal transmission. Figure 1. EPR spectra of S.cerevisiae Nar1.

O036 Pre steady state and steady state kinetic studies into the reaction cycle of the W/Fe/S enzyme formaldehyde oxidoreductase from Pyrococcus furiosus. Emile Bol, Nicolette Broers, Wilfred R. Hagen, Delft University of Technology, Delft, The Netherlands. Contact e-mail: [email protected]

The hyperthermophilic archaeon Pyrococcus furiosus has a highly specific ABC transporter for the internalization of tungstate [1], which it uses to make five tungstoenzymes with a wide range of 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S65

O038 4. Abraham LO, Li Y, Zamble DB. J Inorg Biochem 100: 1005 The S = 3/2 [4Fe-4S]+ cluster of the nitrate reductase A (2006) (NarGHI) from Escherichia coli: Characterisation and implication during the maturation pathway of the O040 complex Pascal Lanciano1, Stéphane Grimaldi1, Alexandra Vergnes2, Structures of intermediates in the oxygen activation Adrien Savoyant1, Axel Magalon2, Patrick Bertrand1, Bruno mechanism of extradiol dioxygenases Guigliarelli1, 1Laboratoire de Bioénergétique et Ingénierie des Elena G. Kovaleva, John D. Lipscomb, University of Minnesota, Protéines, Marseille, France; 2Laboratoire de Chimie Bactérienne, Minneapolis, MN, USA. Marseille, France. Contact e-mail: [email protected] Contact e-mail: [email protected] The homotetrameric enzyme homoprotocatechuate 2,3-dioxygenase Nitrate reductase A from Escherichia coli is a membrane-bound (HPCD) catalyzes proximal extradiol ring-cleavage of catecholic 2+ molybdoenzyme which catalyses the reduction of nitrate to nitrite. substrates. Substrate binding to the active site Fe promotes This respiratory complex is composed of a catalytic subunit (NarG) binding and activation of O2 as well as the subsequent insertion of containing a Mo-bisMGD cofactor (Moco), an electron transfer both oxygen atoms to yield the ring-opened product. It is shown subunit (NarH) carrying four FeS clusters, and a membrane subunit here that the intermediates in the reaction mechanism can be (NarI) with two b-type hemes (1). The resolution of the 3D structure structurally characterized by conducting the reaction in a crystal of of NarGHI has revealed the existence of a fifth FeS cluster (FeS0) HPCD. When the crystal is soaked in the slow substrate 4- with a His-(Cys)3 coordination located close to the Moco (2). In the nitrocatechol (4NC) in a low O2 atmosphere, 3 of the 4 nominally first EPR studies this cluster had escaped detection but recently, low identical subunits in the asymmetric unit contain different intensity EPR lines around g = 5 and typical of a S = 3/2 species intermediates, none of which have been previously characterized were proposed to arise from it (3). A detailed analysis of the EPR [1]. The high-resolution crystal structure shows that these are: (I) a 2+ signal of this S = 3/2 species enabled us to quantify accurately its side-on bound Fe -superoxo-4NC-semiquinone species, (II) an 2+ stoichiometry. In addition, by mutating its amino acid ligands we Fe -alkylperoxo species, and (III) the product complex. Species I assigned definitively the S = 3/2 signal to the FeS0 center. shows that electron density is transferred from the aromatic Moreover we have taken advantage of its peculiar characteristics to substrate to oxygen via the iron, thereby activating both substrates. show that the FeS0 cluster and the Moco are strongly related during This first step in O2 activation may apply broadly in the 2-His-1- the metal insertion processes occurring in the biogenesis pathway of Carboxylate facial triad non-heme iron enzyme family. Intermediate the enzyme (4). In particular, we demonstrate that the sequential II, shows that oxygen attack occurs before O-O bond cleavage and insertion of these two metal centers is orchestrated by a single reveals the position of attack as 4NC-C2. This is the key accessory protein. intermediate in the extradiol dioxygenase mechanism. Similar 1. Blasco, F. et al. (2001) Cell Mol Life Sci 58, 179-93. alkylperoxo species have been proposed for many other 2. Bertero, M. et al. (2003) Nat Struct Biol 10, 681-7. oxygenases, but never observed. Supported by NIHGM24689. [1] 3. Rothery, R. et al. (2004) Biochemistry 43, 5324-33. Kovaleva, E. G., and Lipscomb, J. D. (2007) Science, in press. 4. Lanciano, P. et al. (2007) Manuscript submitted.

O039 DNA-binding properties of Helicobacter pylori NikR Yanjie Li, Deborah B. Zamble, University of Toronto, Toronto, ON, Canada. Contact e-mail: [email protected]

Helicobacter pylori (H. pylori) is a Gram-negative gastric pathogen that is responsible for the majority of peptic ulcer diseases and significantly increases the risk of gastric cancers in humans (1). H. pylori can survive severe acidic shock in the stomach. Nickel, as a cofactor of H. pylori urease and hydrogenase, plays an essential role in H. pylori survival and pathogenesis. H. pylori NikR (HpNikR) is a nickel-responsive transcription factor that controls many O041 important cellular processes such as urease expression and nickel M. uptake either directly or indirectly and was also proposed to be a Catalase-peroxidase mutant KatG[Ser315Thr] from major regulator of the protective response against acid (2). As tuberculosis exhibits unique heme iron structure and HpNikR upregulates or downregulates the expression of different catalytic function despite the remote locus of residue 315. genes (3), the investigation of the DNA-binding affinities of Richard S. Magliozzo1,2, Xiangbo Zhao1, Johannes Schelvis3, HpNikR on various promoters under different conditions is Shengwei Yu1, Kalina Ranguelova1, 1Brooklyn College, Brooklyn, important for understanding the mechanism by which HpNikR NY, USA; 2The Graduate Center, New York, NY, USA; 3New York regulates H. pylori physiological functions. Our previous work has University, New York, NY, USA. shown that HpNikR nickel-dependently binds to different promoters Contact e-mail: [email protected] (4). In this work the binding properties of HpNikR with DNA at different pH values were studied by electrophoresis mobility shift Catalase-peroxidases (KatG) are Class I heme enzymes that exhibit assays and DNA footprinting assays. The results indicate that dual activities. In M. tuberculosis, the organism causing TB, the HpNikR regulates the expression of target genes in a pH-dependent peroxidase activity of KatG is responsible for activation of the old manner. antibiotic isoniazid (INH), leading to formation of an acyl-NAD 1. Dunn BE, Cohen H, Blaser MJ. Clin Microbiol Rev 10: 720 (INH-nicotinamide adenine dinucleotide) adduct regarded as the (1997) bactericidal molecule. The INH-NAD molecule inhibits M. 2. van Vliet AH, Ernst FD, Kusters JG. Trends Microbiol 12: 489 tuberculosis InhA (enoyl-acyl carrier protein-reductase) and (2004) thereby, cell wall (mycolic acid) biosynthesis. The KatG mutant in 3. Contreras M, Thiberge JM, Mandrand-Berthelot MA, Labigne A. which serine 315 is replaced by threonine is responsible for the Mol Microbiol 49: 947 (2003) majority of the world’s INH- resistant TB infections. According to optical, resonance Raman and EPR spectroscopy, KatG[S315T] 123 S66 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

exhibits stable 5-coordinate heme iron in HS and the unusual QS activation by ACC oxidase, steady- and pre-steady-state kinetic spin states, while wild-type KatG, after similar purification and analyses and 18O kinetic isotope effect measurements were brief storage, accumulates 6-c forms. These differences result from employed to determine the order of substrates binding, the nature of a structural change at the heme periphery nearby residue 315. A the activated iron-oxygen species involved in substrate oxidation, biomimetic approach was used to characterize the catalytic and to characterize the intermediates that accumulate during competence of KatG and KatG[S315T] in the activation of INH catalytic turnover. Several ACC substrate analogues that can inhibit under physiologically relevant conditions, where a slow flux of ethylene production have been kinetically investigated with the goal 2 H2O2 (from glucose/glucose oxidase) initiates peroxidase function. of designing a mechanism-based inhibitor. The use of radioactively The mutant enzyme only produced a good yield of INH-NAD in the labeled analogues has been employed to determine the fate of presence of high concentrations of INH, relative to those used substrate breakdown products. Recent detection of radioactive efficiently by wild-type KatG, under conditions where the labeling of specific residues in the active site and subsequent peroxidase intermediates should be produced equally. Isothermal enzyme inactivation suggests an alternative mechanism for titration calorimetry demonstrated low affinity of the mutant substrate oxidation in ACC oxidase. enzyme for INH. The 3-dimensional crystal structures revealed a ACC, H O O narrowed substrate access channel in KatG[S315T] and the origin 2 Asc,O2 His H O His H N of antibiotic resistance due to the steric effects of the methyl group II 2 IV 2 of threonine. Fe Fe Asp H O Asp O 2 DHAsc, His O His H O O042 2 NO-Synthases as a direct source of oxidative and HO nitrative stress. H C=CH , His HN Amandine Maréchal1, Tony A. Mattioli1, Dennis J. Stuehr2, 2 2 FeIII 1 1 Jérôme Santolini , CEA iBiTec-S, Gif-sur-Yvette, France; HCN, CO2 Asp O 2Cleveland Clinic Foundation, Cleveland, OH, USA. His O Contact e-mail: [email protected] (1) Costas, M.; Mehn, M. P.; Jensen, M. P.; Que, L., Jr. Chem. Rev. 2004, 104, 939. Nitric oxide (NO) is a major biological mediator generated in (2) Thrower, J. S.; Mirica, L. M.; McCusker, K. P.; Klinman, J. P. mammals exclusively by proteins from the NO-Synthase family Biochemistry 2006, 45, 13108. (NOS). To explain their implication in both processes of signalling and cytotoxicity, it has been proposed that after NO biosynthesis, NOSs can partition in two distinct catalytic cycles via two different O045 III complexes: i) Fe NO complex whose dissociation leads to NO Purification and structural analysis of Vanadium (signalling) ii) FeIINO complex, whose oxidation produces other reactive nitrogen species (toxicity) with transient formation of Nitrogenase activator in Azotobactor Vinelandii Hiroshi Nakajima1, Takatani Nobuyuki1, Itoh Mitsuko1, Shigetoshi peroxynitrite (PN). Aono2, Yoshihito Watanabe3, 1Nagoya Univ., Nagoya, Japan; To investigate NOSs ability to generate oxidative stress, we studied 2 3 III Institute of Molecular Science, Okazaki, Japan; Nagoya Univ. the model reaction [Fe + PN]. We showed by rapid kinetic Research Center of Materials Science, Nagoya, Japan. measurements that the inducible isoform (iNOS) was activating PN, Contact e-mail: [email protected] enhancing its nitrative potency, with potential damage for biomolecules. This PN activation led to iNOS self-inhibition which Azotobacter vinelandii harbors three genetically distinct could serve as a physiological feedback regulation1. nitrogenases whose expression are regulated by corresponding Lately we investigated the activation of PN by other NOS isoforms, transcriptional activators. VnfA is the activator and known to bind involved in different physiological pathways. Each isoform displays an upstream sequence of vnf operons to express vanadium a distinct PN activation mechanism ranging from toxicity nitrogenase. Previous studies revealed several properties of VnfA enhancement to detoxification. based on in vivo activity assays, while investigation on the Finally, we have been investigating the structure-reactivity molecular structure including a sensing mechanism of an effector relationship of NOS FeIINO complex by combining molecule was thwarted due to difficulty to purify VnfA. In the spectroelectrochemistry, pulsed and classic EPR, and resonance present study, we have achieved purification of wild type VnfA and Raman spectroscopy. We focus on parameters such as the heme preparation of the variants where Cys residues are replaced to structure and environment. Correlated with the kinetic analysis of alanine to inspect the role of Cys residues since Cys rich sequences FeIINO oxidation, these results will lead us to suggest parameters in N-terminus of VnfA, Cys8-X-Cys10-XXXX-Cys15 has been that could promote in situ PN production. suggested to bind any metals or metal clusters to form the sensory This work open new perspectives for the study and the part. understanding of NOSs functioning and biological role. Spectroscopic analysis in vivo and in vitro found the presence of Footnotes 3Fe-4S type clusters ([Fe S ]) in transcriptionally active VnfA, and 1 Maréchal A., Mattioli T.A., Stuehr D.J., Santolini J., J Biol Chem. 3 4 ICP-OES analysis indicated one [Fe S ] in a VnfA monomer. 2007; in press. 3 4 Purified VnfA had a homo-tetrameric form regardless of the presence or absence of [Fe3S4], while VnfA bearing [Fe3S4] showed O043 specific binding affinity to target DNA exclusively, suggesting that [Fe3S4] is responsible to activate VnfA. This is also evidenced by in Mechanistic studies of the ethylene-forming enzyme ACC vivo activity assays in which addition of metal chelater, o- oxidase phenanthroline to growth media of the assay strain decrease the Liviu M. Mirica, Judith P. Klinman, University of California, transcriptional activity of VnfA by 80 %. Mutagenesis analysis Berkeley, CA, USA. supposes Cys10 and 15 serves as ligands of the cluster. Contact e-mail: [email protected]

Our studies have focused on 1-aminocyclopropane-1-carboxylic O046 acid (ACC) oxidase, an ascorbate-dependent non-heme iron enzyme High resolution RX structure of iron(III)-(hydro)peroxo that produces the plant hormone ethylene, important in many aspects of plant growth and development (i.e. germination, fruit intermediates in superoxide reductase SOR 1 ripening, and senescence). In order to study the mode of O2 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S67

Vincent Nivière1, Christelle Mathé1, Tony A. Mattioli2, Chantal O048 3 4 5 Houée-Levin , Catherine Berthomieu , Gergely Katona , Spectroscopic properties and reactivity of a nitric oxide Dominique Bourgeois5, 1iRTSV-CEA/CNRS/Univ. J. Fourier, 2 synthase-like protein from Bacillus subtilis Grenoble, France; iBITEC-S/SB2SM-CEA Saclay/CNRS, Gif-sur- 1 1 1 3 4 Isabelle Salard-Arnaud , Jean-Luc L. Boucher , Daniel Mansuy , Yvette, France; CNRS/Univ. Paris-Sud, Orsay, France; SBVME- Dennis J. Stuehr2, 1Université R. Descartes Paris 5, UMR8601 CEA Cadarache/CNRS/Univ. Aix-Marseille II, Saint-Paul-lez- 2 5 CNRS, Paris, France; Lerner Research Institute, Cleveland, OH, Durance, France; IBS, CEA/CNRS/Univ. J. Fourier, Grenoble, USA. France. Contact e-mail: [email protected] Contact e-mail: [email protected] Mammalian nitric oxide synthases (NOS) are heme-thiolate proteins Superoxide reductase SOR is a novel class of non-heme iron •- that catalyse the oxidation of L-arginine to citrulline and nitric proteins that catalyzes the one-electron reduction of O2 to H2O2, oxide with NȦ-hydroxy-L-arginine (NOHA) as an enzyme-bound providing an antioxidant defense in some bacteria. Its active site 2+ intermediate. Recent genomes sequencing revealed that genes consists of an unusual non-heme Fe center in a [His4 Cys1] square 3+ related to the oxygenase domain of mammalian NOSs exist in some pyramidal pentacoordination, and was proposed to form Fe - gram-positive prokaryotes. peroxide intermediate species during the catalytical cycle. Some of these proteins have been sequenced, cloned and purified. FTIR studies on the SOR from Desulfoarculus baarsii reveal that Recombinant bacterial NOS have been shown to produce nitrite and the protonated carboxylate group of Glu114 is in interaction with citrulline from NOHA and hydrogen peroxide1. In association with the cysteine ligand. Resonance Raman spectroscopy demonstrates a mammalian reductase domain, NOS from Bacillus subtilis is able that mutation of Glu114 into alanine results specifically in a to form nitric oxide in single turnover experiments. Recently, a weakening of the S-Fe bond and increases the extent of freeze- 3+ NOS-like protein from Streptomyces turgidiscabies has been trapping of a Fe -peroxo species after treatment with H2O2 by a implicated in the nitration of a dipeptide phytotoxin required for specific strengthening of the Fe-O bond [1]. plant pathogenicity2,3. However, despite many studies, the role(s) Crystals of the E114A mutant were reacted with H2O2 in order to 3+ and biological function(s) of these prokaryotic NOSs remain(s) trap Fe -(hydro)peroxo species in the crystals. The structure of the unclear. Fe3+-(hydro)peroxo species in the SOR active site was determined 3+ We have performed a spectroscopic study of these new heme- at 1.95 Å resolution. Three different conformations around the Fe - proteins using UV-visible and EPR spectroscopy, and investigated (hydro)peroxo intermediates were characterized. The data their in vitro reactivity. We have demonstrated that a recombinant highlighting the role of a key water molecule finely controlled by NOS from Bacillus subtilis catalyses the transformation of several the enzyme dynamics and reveal the formation of transient hydroxyguanidines to the corresponding ureas and cyanamides and hydrogen bond networks which assist the cleavage of the Fe-O bond the nitration of phenol in a peroxydase catalytic pathway. in order to release the reaction product, hydrogen peroxide [2]. 1 Adak, S., K. S. Aulak, et al. (2002). J Biol Chem 277(18): 16167- 1) C. Mathé, C. O. Weill, T. A. Mattioli, C. Berthomieu, C. Houée- 16171 Levin, E. Tremey, V. Nivière (2007) submitted 2 Kers, J. A., M. J. Wach, et al. (2004). Nature 429(6987): 79-82 2) G. Katona, P. Carpentier, V. Nivière, P. Amara, V. Adam, J. 3 Buddha, M. R., T. Tao, et al. (2004). J Biol Chem 279(48): 49567- Science Ohana, N. Tsanov, D. Bourgeois (2007) , in press 49570

O047 O049 Investigating the role of a highly conserved glutamate in Heme binding to the heme chaperone CcmE reaction intermediate of superoxide reductase Julie M. Stevens1, Takeshi Uchida2, Oliver Daltrop1, Edgar M. 1 1 1 Joao V. Rodrigues , Lígia M. Saraiva , Bruno L. Victor , Harald Harvat1, Teizo Kitagawa3, Stuart J. Ferguson1, 1University of 2 1 3 1 Huber , Cáudio M. Soares , Diane E. Cabelli , Miguel Teixeira , Oxford, Oxford, United Kingdom; 2Hokkaido University, Sapporo, 1 Instituto de Tecnologia Química e Biológica, Oeiras, Portugal; Japan; 3Okazaki Institute for Integrative Bioscience, Okazaki, 2 Lehrstuhl fuer Mikrobiologie der UniVersitaet Regensburg, Japan. 3 Regensburg, Germany; Brookhaven National Laboratory, Upton, Contact e-mail: [email protected] NY, USA. Contact e-mail: [email protected] Cytochromes c are essential metalloproteins found in virtually all organisms and are involved in many fundamental processes Superoxide reductases (SORs) are antioxidant enzymes that including electron transfer in respiratory chains. In E. coli the eliminate the toxic superoxide anion solely by the reductive biogenesis of c-type cytochromes depends on a complex, pathway in contrast with their counterparts, superoxide dismutases multicomponent post-translational system expressed from the (SODs) that catalyze both reduction and oxidation of this radical. In cytochrome c maturation (Ccm) operon ccmABCDEFGH. Central this reaction SOR utilize cellular reductants to convert superoxide to this system is the heme chaperone CcmE which binds heme in into hydrogen peroxide without forming molecular oxygen. These the bacterial periplasm before transferring the cofactor for covalent proteins are associated with anaerobic lifestyle since they are found attachment to apocytochromes c. CcmE has been found to have only in anaerobic or microaerophilic prokaryotes, and, in many unusual properties including covalent binding via a histidine residue cases where the canonical SODs are absent, they constitute the only to a heme vinyl group [1,2], tyrosine ligation of the heme iron [3], a known protecting system against superoxide. change in coordination depending on the iron oxidation state [4] The reaction mechanism of SOR from the hyperthermophilic involving residues in the flexible C-terminal domain of the protein archaeon Archaeoglobus fulgidus was previously studied by pulse [5]. We present results on these phenomena and discuss their radiolysis, allowing the observation of a putative ferric- implications. hydroperoxide intermediate, which decayed upon protonation to [1] Daltrop O et al. (2002) PNAS 99:9703 produce hydrogen peroxide. We also demonstrated the role of [2] Stevens JM et al. (2003) J Biol Chem 278:20500 3+ glutamate (E12) in the protonation of the Fe -OOH intermediate; [3] Uchida T et al. (2004) J Biol Chem 279:51981 this residue also functions as the sixth ligand to the oxidized iron [4] Stevens JM et al. (2006) J Biol Chem 281:6144 center. The glutamate ligand is highly conserved among known [5] Harvat EM et al. (2005) J Biol Chem 280:36747 SORs with only a few exceptions. Here we study by pulse radiolysis the reduction of superoxide by a natural glutamate-lacking “mutant”, and show that this enzyme is fully functional. This suggests that the enzyme has evolved divergently to be able to efficiently protonate the intermediate in the absence of a glutamate. 123 S68 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

O050 The 3-His ligated nonheme Fe(II)-centre in Diketone Dioxygenase Dke1: probing the metallocentre's intrinsic reactivity and the role of the metal ion's second coordination sphere in catalysis. Grit D. Straganz, Sigrid Egger, Bernd Nidetzky, Graz University of Technology, Graz, Austria. Contact e-mail: [email protected]

Diketone cleaving dioxygenase Dke1, which has been isolated from Acinetobacter johnsonii, shows unique catalytic properties, oxygenatively cleaving the C-C bond of enolizable beta-diketones to the corresponding acid and alpha-oxo aldehyde. Its active site shows an atypical nonheme Fe(II) centre, which is not ligated via the ‘classical’ 2-His-1-carboxylate motif but via a facial triad of three histidines (1). The substrate's electronic properties govern the rate of dioxygen reduction and results from quantitative structure- activity relationship analysis lead to the proposal of a chemical mechanism of diketone cleavage in Dke1 (2). But what role does the protein environment play in catalysis? O052 Investigating the impact of the metal’s second coordination sphere The crystal structure of cytochrome P460 of on particular steps of catalysis by mutational and kinetic analysis, Nitrosomonas europaea reveals a novel cytochrome fold we identified a gate constituted of three amino acid residues that and heme-protein cross-link. promotes diketone binding and oxidation. A basal reactivity of the Carrie M. Wilmot1, Arwen R. Pearson1, Bradley O. Elmore1, metallocentre towards 'alien' (non-beta-dicarbonyl) substrates, on Cheng Yang2, Joseph D. Ferrara2, Alan B. Hooper1, 1University of the other hand, is apparently insensitve towards alterations in the Minnesota, Minneapolis, MN, USA; 2Rigaku Americas Corp., The metal’s second sphere, and demonstrates the intrinsic reactivity of Woodlands, TX, USA. the 3-His metallocentre towards 'typical' dioxygenase substrates. Contact e-mail: [email protected] Taken together, our findings give interesting insights into the interplay of metallocentre, protein environment and substrate We have determined the 1.8 Å X-ray crystal structure of a mono- structure in Dke1. heme c-type cytochrome, cytochrome P460, from Nitrosomonas (1) (a) Straganz G. D., Glieder A., Brecker L., Ribbons D.W., europea. The chromophore possesses unusual spectral properties Steiner W. Biochem. J. 369 (2003) 573-581. (b) Stranzl G. R. PhD analogous to those of the catalytic heme P460 of hydroxylamine Thesis (2002) Karl Franzens University Graz, Austria. oxidoreductase (HAO), the only known heme in biology to (2) Straganz G. D., Nidetzky B. J. Am. Chem. Soc. 127 (2005) withdraw electrons from substrate coordinated to the iron. The 12306-12314. (b) Straganz G.D., Hofer H., Steiner W., Nidetzky B. analysis reveals a homodimeric structure and elucidates a new c- J. Am. Chem. Soc. 126 (2004), 12202-12203. type cytochrome fold that is predominantly ȕ-sheet. In addition to the two cysteine thioether links to the porphyrin typical of c-type hemes, there is a third proteinaceous link involving a conserved O051 lysine. The covalent bond is between the lysine side-chain nitrogen Monitoring the redox state of the laccase type-1 and Cu3 and the 13'-meso carbon of the heme, which following cross-link copper centres by fluorescence formation is sp3 hybridized demonstrating loss of conjugation at this Armand W. J. W. Tepper1, Florien G. de Brouwer1, Erik position within the porphyrin. The structure has implications for the Vijgenboom1, Thijs J. Aartsma2, Gerard W. Canters1, 1Leiden analogous tyrosine-heme meso carbon cross-link observed in HAO. Institute of Chemistry, Leiden, The Netherlands; 2Leiden Institute of Physics, Leiden, The Netherlands. Contact e-mail: [email protected]

Many redox active cofactors change color upon changing oxidation state or the binding of a ligand. For this reason, UV-Vis spectroscopy is a principal technique in studying the structure and function of redox proteins. Its downsides are the low sensitivity and selectivity. To circumvent these problems, we have used fluorescent labeling to translate these absorption changes into a fluorescence signal with the benefits of enhanced selectivity combined with single-molecule sensitivity 1. Here we report on an extension of this methodology to study redox processes in SLAC, a multicopper laccase from S. coelicolor. It couples the four electron reduction of dioxygen with the oxidation of small organic substrates. Laccases contain two chromophores: a blue type-1 copper site, which accepts electrons from reducing substrate, as well as a green trinuclear Cu3 copper cluster, which is the site of O2 reduction (see figure). The optical absorption of these centers depends on their oxidation state. We show that the redox-state of the type-1 site and the Cu3 cluster can be simultaneously and independently monitored by following the emission of two different fluorescent reporters. Several examples illustrating the technique as well as potential applications will be discussed. 1 S. Kuznetsova, G. Zauner, R. Schmauder, O. A. Mayboroda, A. M. Deelder, T. J. Aartsma, G. W. Canters. Analytical Biochemistry 2006, 350(1), 52-60. 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S69

limits of detection and of quantitation of ascorbic acid has been reached. The sensitivity of the method was higher for Trametes versicolor than for Agaricus bisporus laccase. The test is sensitive, precise and quick and requires low quantities of sample. The method has been applied to assays of quality control in drugs that contain as active ingredient. Furthermore, the method is useful to evaluate the antioxidant activity of ascorbic acid. The assay does not require electrochemical neither spectrofluorimetric instruments. The method is applicable on spectrophotometers (1mL cuvette) and on multiwell microplate readers, which favours its mechanization for high throughput screen assays. The authors are grateful to NOVOZYMES SPAIN S.A. by free samples of laccase. This work has been partially supported by grants from several Spanish organizations. (J.T.) Projects 00672/PI/04 Fundacion Seneca and BioCARM BIO-BMC O053 06/01/0004, CARM, Murcia; as well as Project BIO2006-15363, The Role of SlyD in Nickel Incorporation in Escherichia MEC, Madrid. (R.M.G.) NOVOZYMES SPAIN S.A. (M.A.) Doctorate fellowship BES-2004-3829, FPI-MEC, Madrid. coli [NiFe] Hydrogenase Biosynthesis Pathway Jie Wei Zhang, Deborah Zamble, University of Toronto, Toronto, ON, Canada. Contact e-mail: [email protected] P100 Rubredoxin as a paramagnetic shift-inducing probe E.coli expresses at least three [NiFe] hydrogenase enzymes, which Rui M. Almeida, Sofia R. Pauleta, Isabel Moura, José J. G. Moura, catalyze the production or consumption of hydrogen gas and occupy REQUIMTE/CQFB, Caparica, Portugal. a central place in the energy metabolism of anaerobic bacteria1. The Contact e-mail: [email protected] assembly of the hydrogenase metallocenters requires SlyD and the proteins encoded by the hyp genes (hypABCDEF) [1, 2]. SlyD is a Rubredoxin (Rd) is a small protein (~5 kDa), containing a simple member of the FK-506 binding of Peptidyl-prolyl redox centre (one iron atom coordinated to four cysteinyl residues), (PPIase) [3]. In addition to its PPIase domain, SlyD is present in all species of the Desulfovibrio genus. Rd has been composed of a molecular chaperone domain and a C-terminal tail assigned to play an important role in the oxygen detoxification rich in potential metal binding residues. SlyD directly interacts with pathway, donating electrons to enzymes such as superoxide HypB and the disruption of the SlyD-HypB complex results in reductase and rubredoxin:oxygen oxido-reductase. In the oxidized deficient hydrogenase production in vivo [2, 4]. form, the inherent high-spin of the Fe(III) ion can be used to probe To examine the role of SlyD in hydrogenase biosynthesis, mutations the interaction site of other metalloproteins, in particular using that disrupt the PPIase activity were prepared. SlyD(I42S, F132Y) NMR methods, due to the induced pseudocontact shift and and SlyD(I42S), which displayed 1% and 40% of wild-type levels linewidth broadening. In this study, we used Desulfovibrio gigas Rd of PPIase activity respectively, could restore 61% ±10 and 41% ±9 (both Fe and Zn-substituted forms) and cytochrome c3, a tetraheme of total hydrogenase activity in vivo. Both mutants interacted with containing protein, isolated from the same organism, as a model of HypB in vitro and accumulated the same level of cellular nickel a transient electron transfer complex. In order to determine these under anaerobic growth condition. These results suggest that the effects we have used 1H NMR titrations. We have observed that PPIase activity of SlyD does not contribute to cellular nickel increasing amounts of rubredoxin induce selective heme methyl accumulation or hydrogenase activity. Additional in vivo shifts and linewidth broadenings (only with the Fe form), and an experiments suggest that the SlyD-HypB complex may play a role apparent Kd of ~10 ȝM was determined. These results are in in nickel storage in E.coli. agreement with the higher ranked complexes obtained using a soft- 1. Casalot L., et al., Trends Microbiol, 2001, p228. docking algorithm program, BiGGER1, that predicted a cluster of 2. Zhang, J.W., et al., J Biol Chem, 2005, p4360. solutions with Rd binding near the heme IV of cytochrome c3. 3. Roof, D.W., et al., FEMS Microbiol Rev, 1995, p213. [1] Palma PN, Krippahl L, Wampler JE, Moura JJG (2000) Proteins 4. Leach, M.R, et al., (manuscript submitted 2007). 39, 372 Ackowledgements: R.M.A. and S.R.P. wish to thank Fundação para a Ciência e Tecnologia for PhD (SFRH/BD/25342/2005) and pos- POSTERS doc (SFRH/BPD/14067/2003) grants, respectively.

P099 P101 Spectrophotometric determination of ascorbic acid with laccase oxidizing ABTS Biochemical and catalytic characterization of Myriam Abad1, Virginia Tomas2, Ramiro Martinez-Gutierrez3, Desulfoviridin from Desulfovibrio desulfuricans ATCC Francisco Garcia-Canovas1, Jose Tudela4, 1GENZ-Grupo de 27774 - towards the development of a sulfite biosensor investigacion Enzimologia, Universidad de Murcia, Spain; 2Dept. Alexandra S. Serra1, Isabel Moura1, José J. G. Moura1, Maria G. Quimica Analitica, Universidad de Murcia, Spain; 3NOVOZYMES Almeida1,2, 1REQUIMTE/CQFB, Dept. Quimica, Faculdade de Spain S.A., Madrid, Spain; 4GENZ (http://www.um.es/genz), Ciências e Tecnologia, Monte Caparica, Portugal; 2Escola Superior Universidad de Murcia, Spain. de Saúde Egas Moniz, Monte de Caparica, Portugal. Contact e-mail: [email protected] Contact e-mail: [email protected]

Laccase (EC 1.10.3.1) is a four copper oxidase that catalyze the Thanks to their antimicrobial and antioxidant activities, sulfur oxidation by molecular oxygen of phenols up to phenoxyl radicals, dioxide and sulfites have been used as food additives for many which can evolve up to quinones. These are reduced by ascorbic years. However, sulfites have been occasionally implicated as acid (vitamin C) which oxidizes to dehydroascorbic acid. The asthma triggers [1]. As a consequence, the EC Directive 95/2/EC spectrophotometric assays of laccase oxidizing ABTS in presence includes sulfites in the list of preservatives and antioxidants of ascorbic acid, they show a lag period proportional to the endorsed in specified products with a Maximum Permitted Level concentration of this vitamin. The assay conditions, reaction [2]. The classical methods for sulfite determination in food products medium, concentration of enzyme and of chromogenic substrate are time consuming and have a low sensitivity. Therefore, is very have been optimized. A broad linear range with submicromolar important to develop a new analytical tool capable of easily 123 S70 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

quantifying sulfites in complex samples. With the aim of attachment motif, CXXCH), a large proportion of which contain constructing an electrochemical sulfite biosensor based on a multiple heme groups. This profusion of cytochromes confers upon selective enzyme, we have been studying and characterizing the the organism a wide respiratory flexibility and the ability to utilise a dissimilatory sulfite reductase from Desulfovibrio desulfuricans diverse range of electron acceptors, including Fe(III) and Mn(IV), ATCC 27774 - Desulfoviridin (Dsv) The enzyme is composed by nitrite, nitrate, thiosulfate, sulfate and fumarate. three subunits of ca. 50, 40 and 11 kDa, all of them showing a A putative octaheme cytochrome, containing eight CXXCH motifs, strong homology to Dsv from Desulfovibrio vulgaris at the N- with no homologs of known function has been identified and terminal. The iron content, as determined by ICP-AES was slightly overexpressed. This protein has been purified and identified as a lower than expected (17±4). From the biosensor point-of-view, the tetrathionate reductase, and the structure has been solved to 2.2 low specific activity of Dsv (nmolmg-1min-1 range) and the Å.[2],[3] Despite the presence of eight conventional heme- instability of sulfites in solution constitute important drawbacks that attachment motifs, the protein structure revealed the presence of should be overcome. In this regard, we also examined by cyclic seven normally ligated c-type heme groups and one unusual lysine- voltammetry the ability of a set of electronic mediators alternative ligated CXXCK heme at the active site. This unconventional lysine to methyl viologen (phenosafranine, meldola blue/NADH, neutral heme ligation has been previously observed only in the pentaheme red, benzyl viologen and bromophenol blue) to efficiently shuttle nitrite reductases, however in these cases lysine ligation was electrons to Dsv. predicted by a CXXCK motif. 1- Taylor SL; Higley NA; Bush RK (1986) Adv Food Res 30, 1-76 Assays to determine potential substrates for this enzyme have 2- EC Official Journal (1995) L61, 1-53 shown activity with a range of nitrogen-containing compounds, including nitrite, nitric oxide, nitrous oxide and hydroxylamine. Additionally, a mutant of the ligating lysine has been prepared and P102 work is currently underway to determine its structure and kinetics. Novel non-heme iron lipoxygenases from cyanobacterium [1] Heidelberg, J.F. et al. (2002) Nat. Biotechnol. 20, 1118-1123. Nostoc punctiforme [2] Rothery, E.L. (2004) Ph.D Thesis, University of Edinburgh Alexandra Andreou, Imke Lang, Ivo Feussner, Dept. of Plant [3] Mowat, C.G. et al. (2004) Nat. Struct. Biol. 11, 1023-1024. Biochemistry, Georg-August-University of Göttingen, Albrecht- von-Haller-Institute for Plant Sciences, Göttingen, Germany. Contact e-mail: [email protected] P104 Investigation of the catalytic cycle of laccase immobilized Lipoxygenases (LOXs) are a family of structurally related non- on gold electrodes heme iron containing dioxygenases. They catalyse the insertion of Véronique Balland1, Christelle Hureau1, Erin Wallace2, Thierry molecular oxygen into polyunsaturated fatty acids that contain one Tron2, Benoit Limoges1, 1Laboratoire d'Electrochimie Moléculaire, or more 1,4 pentadiene moieties to give the corresponding CNRS UMR 7591, Université Paris Diderot, Paris, France; hydroperoxides. LOX products can be further metabolized to yield 2Laboratoire BiosCiences, FRE3005, Université Paul Cézanne, signaling molecules such as aldehydes and jasmonate. The insertion Marseille, France. of oxygen exhibits high regio- and stereo-specificity. This Contact e-mail: [email protected] specificity is dependent on the primary sequence of the enzyme, which is predicted to determine the orientation and depth of Laccases are four-copper containing enzymes exhibiting dual substrate penetration into the active site. reactivity. Their ability to oxidize a large panel of substrates Although there is an abundance of information on plant and animal including recalcitrant environmental pollutants and to reduce LOXs, the number of prokaryotic LOXs that have been described is molecular dioxygen into water makes them promising enzymes for limited. Prokaryotic and eukaryotic algae are known as sources of the development of biosensors and biofuel cells, respectively. biologically active compounds among which are alcohols, alkanes, However, these applications require the challenging preparation of aldehydes and branched hydrocarbons, which may derive from stable enzymes-coated electrodes with well-defined catalytic primary LOX products. Analysis of the genomic sequence of the properties toward both substrates and dioxygen. cyanobacterium Nostoc punctiforme PCC 73102 revealed two We report the immobilization of a recombinant Trametes C30 genes with homology to the LOX of the prokaryotic organism LAC3[1] using the nitrilotriacetic acid (NTA)/histidine-tag Pseudomonas aerigunosa. The two enzymes, NpLOX1 and technology that requires mild protein modification. Analysis of the NpLOX2, were isolated and tested for catalytic activity against his-tagged LAC3 in solution indicates that addition of the linoleic and Į-linolenic acid. Both proteins were identified as polyhistidine moiety does not strongly affect its catalytic activity. linoleate 13-LOXs by expression in E. coli. Interestingly, at the Moreover, it allows formation of a stable enzyme monolayer on position of the postulated determinants of regiospecificity within the gold electrodes. The catalytic cycle of immobilized LAC3 was active site, the two enzymes contain different amino acids in investigated by cyclic voltammetry and analysed using available comparison to known 13-LOXs. NpLOX1 was further theory for ping-pong mechanisms and immobilized enzymes.[2] To characterized: It showed a broad pH optimum ranging from pH 4.5 achieve a complete analysis of LAC3, the kinetic characteristics of to pH 8.5 with a maximum at pH 8.0 and Į-linolenic acid was the the immobilized enzyme were compared with their homogeneous preferred substrate. counterparts. Our results indicate that high surface coverage of fully functional LAC3 can be obtained by using this immobilization procedure. P103 Octaheme tetrathionate reductase (OTR) is involved in the reactions of the nitrogen cycle Sally J. Atkinson1, Vivek A. Jassal1, Emma L. Rothery1, Chris G. Mowat1, Caroline S. Miles2, Graeme A. Reid2, Stephen K. Chapman1, 1University of Edinburgh, Edinburgh, United Kingdom; 2Institute of Cell and Molecular Biology, Edinburgh, United Kingdom. Contact e-mail: [email protected]

Shewanella oneidensis MR-1 is a gram-negative Ȗ-proteobacterium isolated from the freshwater Lake Oneida, USA. Genomic analysis of this metal-reducing bacterium has identified 39 genes encoding c-type cytochromes[1] (determined using the c-type heme 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S71

polypeptide that binds a modified heme via two ester linkages. By contrast, MPO is a dimeric protein with a single disulfide bridge between symmetry-related halves, each of which contains two polypeptides. In MPO the heme is also covalently attached to the protein via two ester bonds, but it is the only heme protein with an additional sulfonium ion linkage. The mode of heme binding strongly affects the biophysical and biochemical properties of these metalloproteins. In order to elucidate the effect of heme to protein linkages on the thermal and conformational stability of LPO and MPO in their ferric forms, we have performed comparative electronic absorbance, electronic circular dichroism and intrinsic steady-state fluorescence studies on protein unfolding by urea and guanidine hydrochloride as well as with increasing temperature both in the absence and presence of dithiothreitol. Determination of thermodynamic (free enthalpies, enthalpies and entropies) as well as kinetic parameters of denaturation revealed differences between LPO and MPO, which will be discussed with respect to the known structures of human MPO and bovine LPO.

P107 Delineating the interface between sMMO components. Rahul Banerjee, Jingyan Zhang, Codrina Popescu, John D. [1] Klonowska A., Gaudin C., Asso M., Fournel A., Reglier M., Lipscomb, University of Minnesota, Minneapolis, MN, USA. Tron T. (2005) Enzyme and Microbial Technology 36, 24-41 Contact e-mail: [email protected] [2] a- Andrieux C.P., Limoges B., Marchal D., Savéant J-M. (2006) Anal. Chem. 78, 3138-43. b- Limoges, B, Moiroux, J, Savéant, J.- Soluble utilizes a bis-ȝ-oxo diiron(IV) M. (2002) J. Electroanal. Chem. 521, 8. intermediate ‘Q’ in the buried active site of the hydroxylase component (MMOH) to oxidize methane. However, an accessory protein (MMOB) is required to regulate the reaction of substrates P105 with Q. MMOB forms a specific complex with MMOH that Biochemical Characterization of Human COX11 controls access to Q on the basis of substrate size. This allows Lucia Banci, Ivano Bertini, Adele Fantoni, Sayaka Inagaki, MMO to preferentially oxidize methane in the presence of other Magnetic Resonance Center CERM, Florence, Italy. potential substrates with weaker C-H bonds. The binding of MMOB Contact e-mail: [email protected] perturbs the spectroscopic characteristics of the diiron cluster as well as the extent to which the C-H bond breaking reaction COX11 is a copper-binding protein, which plays a crucial role in proceeds by quantum tunneling. Thus, the effects of the surface the incorporation of copper into the CuB site of cytochrome c interaction are transmitted to the MMOH active site. Using NMR oxidase. In this study, we carried out the expression and relaxation and site directed spin-labeling, we have mapped the characterization of human COX11 (hCOX11). The gene of interface between the MMOH and MMOB components [1]. There hCOX11 codes 276 amino acids, parts of which are predicted to be are clear solvent exposed and buried MMOB surfaces in the present as trans-membrane helices. We cloned C-terminal soluble complex, but the relative orientation of the two components could domain, Cys121-Asn276, in pPICZ B vector. The expression not be determined by these approaches. In current studies, plasmid was used to transform into the methylotrophic yeast Pichia fluorescence donor-acceptor pairs are placed on MMOH and pastoris (P. pastoris) X-33. The hCOX11 was expressed as a MMOB so that the specific orientation of the two components can soluble form in P. pastoris under control of the inducible alcohol be mapped using FRET techniques. Supported by NIHGM40466. oxidase 1 promoter. The purified hCOX11 was observed at ~ 18 [1] Zhang, J.; Wallar, B., J.; Popescu, C., V.; Renner, D., B.; kDa on SDS-PAGE. This result is consistent with the molecular Thomas, D., D.; Lipscomb, J. D. Biochemistry 2006, 45, 2913-2926 weight calculated from the translated cDNA sequence. The far-UV circular dichroism spectrum of hCOX11 showed characteristic features of a folded protein with beta-strands and alpha-helical structures. Analytical gel filtration showed that hCOX11 was eluted as dimmer with no effect induced by the reducing agent DTT. This behavior suggests that the dimerization would not involve formation of disulfide bonds. The mechanisms responsible of the dimerization and the interaction with copper are under study. The results will be present and discussed.

P106 Thermal and conformational stability of lactoperoxidase and myeloperoxidase Srijib Banerjee, Paul Georg Furtmüller, Christian Obinger, Department of Chemistry, Division of Biochemistry, BOKU - University of Natural Resources and Applied Life Sciences, Vienna, Austria. Contact e-mail: [email protected]

Lactoperoxidase (LPO) and myeloperoxidase (MPO) belong to the peroxidase- superfamily. Both enzymes are critical elements of the innate, i.e. nonantigenic immune system. Lactoperoxidase is a monomeric protein consisting of a single 123 S72 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

P108 P110 Subunits Identification of the Complex I from Conformational control of S100A2 by metal ions Rhodothermus marinus Hugo M. Botelho1, Michael Koch2, Günter Fritz2, Cláudio M. Ana P. Batista, Andreia S. Fernandes, Ana Coelho, Manuela P. Gomes1, 1Instituto de Tecnologia Química e Biológica, Oeiras, Pereira, Miguel Teixeira, Instituto de Tecnologia Química e Portugal; 2Department of Biology, University of Konstanz, Biológica, Oeiras, Portugal. Konstanz, Germany. Contact e-mail: [email protected] Contact e-mail: [email protected]

Complex I, NADH:quinone oxidoreductase, is the first and the The S100 proteins are small, dimeric Ca2+-binding proteins biggest complex of all that constitute respiratory chain. This involved in cell cycle regulation and cell growth, differentiation and enzyme couples electron transfer from NADH to quinone to the mobility in vertebrates. Several studies indicate expression proton translocation across the membrane, which contributes to the misregulation of these proteins in a number of cancers. The Ca2+ membrane electrochemical potential that is the driving force for binding sites are EF-hands. As in other EF-hand proteins, Ca2+ ATP synthesis. Complex I is composed of multiple polypeptide binding to S100A2 elicits the displacement of an helix exposing a subunits and contains several prosthetic groups involved in its hydrophobic patch which is the docking site for proteins involved in activity. The fact that many mitochondrial diseases are related to downstream signalling. The most studied one is transcription factor complex I defects shows the need to know more about the structure p53. and function of this large respiratory chain complex. Rhodothermus Besides binding Ca2+, S100A2 also binds Zn2+ with high affinity. marinus complex I, our model system, is a NADH:menaquinone Each S100A2 monomer binds two Zn2+ ions in sites different from oxidoreductase and has been extensively characterized [1,2]. The the Ca2+ ones. The physiological role of Zn2+ seems to be the genes coding for R. marinus Nqo subunits are clustering in two modulation of the Ca2+ affinity, as Zn2+ binding to one of the sites 2+ operons, nqo1-7 (nqoA) and nqo10-14 (nqoB) and two independent decreases the binding affinity of the Ca , a unique mechanism in genes, nqo8 and nqo9.[3] Our recent results concerning the the S100 family so far. identification of the subunits are reported. By mass spectrometry, of Our current research goal is to characterize the conformational and the complex in solution, was possible identified all the subunits of stability properties of S100A2 discriminating the effect of the the complex. Using this same technique, with digest SDS-PAGE binding of each metal to its cognate site. To accomplish this, bands, were identified just the subunits that constitute the peripheral engineered forms of S100A2 with mutated Zn2+ cysteine ligands part of the complex, Nqo1-5 and Nqo9. The subunits Nqo1, Nqo3 were used. Biophysical and biochemical techniques such as circular and Nqo9 were also identified by immunoblotting with antibodies dichroism and fluorescence spectroscopies have been employed in against subunits of Neurospora crassa Complex I.[1] assessing conformational changes dependent of temperature, [1] A.S. Fernandes, M.M. Pereira and M. Teixeira, J. of Bioenerg. chemical denaturant concentration and metal loading status. Here and Biomemb. 2002, 34, 413-421. we report the effects of Zn2+ binding to the high affinity site and [2] A.S. Fernandes, F.L.Sousa, M. Teixeira and M.M.Pereira, discuss its repercussions in the physiology and conformational Biochemistry 2006, 45(3), 1002-8. tuning of S100A2. [3] A.M.P.Melo, et al, Biochimica et Biophysica Acta. 2005, 1709, This work is supported by FCT/MCTES, Portugal. HMB is 95-103. recipient of a Ph.D. fellowship (FCT/MCTES SFRH/BD/31126/2006).

P109 Cyanobacterial terminal heme-copper oxidases and their P111 electron donors Synthesis and evaluation of new amino-acids as arginase Margit Bernroitner1, Doris Gusenbauer1, Günter A. Peschek2, inhibitors Christian Obinger1, 1BOKU - University of Natural Resources and Jean-Luc Boucher1, David Christianson2, Sylvie Dijols1, Yves Applied Life Sciences, Vienna, Austria; 2University of Vienna, Frapart1, Stephanie Pethe1, Daniel Mansuy1, 1CNRS-UMR 8601, Vienna, Austria. Paris, France; 2Roy and Diana Vagelos Laboratories, University of Contact e-mail: [email protected] Pennsylvania, Philadelphia, MA, USA. Contact e-mail: [email protected] Photosynthetic (PET) and respiratory electron transport (RET) are physically separated in eukaryotic cells but share common In mammalian cells, L-arginine is metabolized by two major components (e.g. plastoquinone, cytochrome b6f, cytochrome c6, pathways: arginases catalyze its hydrolysis to L-ornithine and urea, plastocyanin etc.) in cyanobacteria. As in most other phototrophic whereas NO synthases (NOSs) catalyze its oxidation to L-citrulline prokaryotes, the intracytoplasmic membrane contains a dual- and nitric oxide, NO. NO is an important biological molecule function PET-RET system, whereas the cytoplasmic membrane involved in vasodilation, neurotransmission and immune responses. contains only RET without photosynthetic reaction centres. In RET Recent studies support the hypothesis that arginases may be terminal respiratory oxidases (TROs) function as the final electron essential in the regulation of NOSs activities by modulating local L- acceptor (“electron sink”) reducing O2 to water in a concerted four- arginine concentration and it is observed that inhibition of arginases electron transfer reaction. Genome and expression analysis suggest led to an enhancement of NO production. The search for potent that cyanobacteria can contain one or more representatives of TROs inhibitors of arginases is thus the focus of active studies. from both the heme-copper oxidase superfamily and the non-copper L-Arginine hydrolysis by arginases is achieved by a metal-activated II or bd-type oxidase superfamily. A detailed genomic (29 strains, 19 water molecule that bridges a (Mn )2 cluster at their active site. The completely sequenced genomes), sequence and structural (subunits I hydrolysis is postulated to proceed through a tetrahedral II & II) analysis of cyanobacterial heme-copper oxidases and their intermediate resulting from nucleophilic attack of a (Mn )2-bound putative electron donor proteins (cytochrome c6, plastocyanin and hydroxide ion at the guanidinium carbon of L-arginine. Using X-ray cytochrome cM) revealed that heterocyst-forming nitrogen-fixing data and a model proposed to explain the inhibitory effects of L- cyanobacteria usually have more than one gene for obligatory arginine analogues, we have synthesized and tested new amino- (aa3-type) and cytochrome c6. Many acids as putative inhibitors of rat liver arginase. EPR spectroscopy II cyanobacterial strains lack genes encoding heme copper quinol has been used to study their positionings at the (Mn )2 active site. oxidases. All cyanobacteria have a single-copy gene for cytochrome The present poster will resume results obtained in our search for cM and plastocyanin (exceptions Gloebacter violaceus & arginase inhibitors. Thermosynechococcus elongatus). These data are discussed with respect to known in vitro interactions between these metalloproteins and to physiological relevance. 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S73

P112 P114 Biochemical and spectroscopic analysis of a Orange proteins from Desulfovibrio gigas and bacteriophytochrome from Pseudomonas aeruginosa Desulfovibrio alaskensis Latifa Bouzhir, Marten H. Vos, Ursula Liebl, INSERM U696- Marta S. Carepo1, Americo Duarte1, Sofia R. Pauleta1, Anthony G. CNRS UMR7645, Ecole Polytechnique, Palaiseau, France. Wedd2, Alice S. Pereira1, Jose J. G. Moura1, Isabel Moura1, Contact e-mail: [email protected] 1Requimte – Departamento de Quimica, CQFB, Faculdade de Ciências e Tecnologia – UNL, Monte da Caparica, Portugal; 2School of Chemistry, University of Melbourne, ParkVill, Australia. Bacteriophytochromes (BphPs) are bacterial photoreceptors that Contact e-mail: [email protected] sense red/far-red light and bind a biliverdin chromophore, an immediate breakdown product of heme (1). Bacteriophytochromes The orange protein (ORP) isolated from Desulfovibrio gigas has a 3+ are known to regulate the biosynthesis of photosynthetic and mixed-metal sulphide cluster of the type [S2MoS2CuS2MoS2] nonphotosynthetic pigments and motility, but have a wider array of (1).The protein is a monomer of 11.8 kDa and the cofactor functions, only partially known. Here we describe the biochemical contributes to a characteristic Uv-visible spectra with maximum and spectroscopic analysis of a novel bacteriophytochrome, absorption peaks at 480 and 338 nm (2). The ORP was PA5442, from the opportunistic pathogen Pseudomonas aeruginosa. heterologously expressed in E. coli as an apo-protein. In this work 2- The protein is composed of a PAS domain linked to a GGDEF and we report the reconstitution of the Mo-Cu cluster using [MoS4] EAL output module that is involved respectively in synthesis and CuCl2 in the over-expressed apo-protein. Biochemical (diguanylate cyclase) and degradation (phosphodiesterase) of the characterization of the reconstituted protein was performed bacterial second messenger c-di-GMP. GGDEF-EAL-containing including Uv-visible spectra, metal content and EPR spectroscopy proteins studied to date displayed either diguanylate cyclase or and the results were compared with the native protein. We are also phosphodiesterase activity, but not both. To get insight into the presenting a preliminary biochemical characterization of an orange function of PA5442, the protein was expressed heterologously in protein isolated from Desulfovibrio alaskensis. The metal content Escherichia coli and purified in the dark using affinity ratio determined by ICP for this protein is 2 Mo to a 1 Cu as found chromatography. Recombinant PA5442 has an apparent molecular for D.gigas. The protein is also a monomer of approximately 12.8 mass of 106 kDa and shows the characteristic red/far-red-light- kDa. The N-terminal of the D. alaskensis ORP was chemically induced photoreversibility of bonafide phytochromes. Steady-state sequenced and the NCBI alignment revealed an identity of 90 % absorption spectroscopy possibly indicates a bound heme, although homology with a MTH1175-like domain family protein predicted to date it is not known if it is an intrinsic component of PA5442 and by Desulfovibrio desulfuricans G20 complete genome sequencing essential for its role in vivo. and annotation. (1) Bhoo SH, Davis SJ, Walker J, Karniol B, Vierstra RD (2001) (1) 1. George, G.N. Pickering, I. J., Yu, E. Y., Prince, R.C. Nature 414, 776 Bursakov SA, Gavel O.Y. Moura J.J.G, and Moura I,JACS, 2000, 122, 8321-8322. (2) Bursakov S.A., Gavel O.Y., Di Rocco G., Lampreia J., Calvete P113 J., Pereira A.S., Moura J.J.G., and Moura I, J Inorg Biochem, 2004, CO-inhibition of NiFe hydrogenases monitored by EPR 98(5), 833-840. spectroscopy Marta S. Carepo is supported by Grant SFRH/BPD/24150/2005- Bénédicte Burlat, Fanny Leroux, Patrick Bertrand, Sébastien POCTI Dementin, Marc Rousset, Christophe Léger, Bruno Guigliarelli, Laboratoire de Bioénergétique et Ingénierie des Protéines, Marseille, France. P115 Contact e-mail: [email protected] Naphthalene dioxygenase-catalyzed cis-dihydroxylation of substituted 2-pyridones and 2-quinolones Hydrogenases are enzymes which catalyze the reversible oxidation Claude Chopard, Robert Azerad, UMR 8601 CNRS Univ. René of dihydrogen into protons and electrons. D. fructosovorans NiFe Descartes, Paris, France. hydrogenase consists of two subunits and contains a nickel-iron Contact e-mail: [email protected] active site and three iron-sulfur clusters involved in electron transferring. Hydrophobic channels (1) connect the NiFe active site The bacterial metabolism of aromatic compounds often proceeds (deeply buried in the protein) to the surface of the protein and via an initial dioxygenase-catalyzed dihydroxylation to yield cis- enables diffusion of substrate H2 and small inhibitors (O2, CO) dihydrodiol derivatives. A large range of mono- and polycyclic towards the active site. substrates have been reported to produce the corresponding In this study, we focused on inhibition by CO of the NiFe active site enantiopure cis-dihydrodiol metabolites. However, to date, only a by using EPR spectroscopy. From the so-called paramagnetic Ni-C few data have been reported about the cis-dihydroxylation of species corresponding to an intermediate state on the catalytic compounds derived from heterocyclic arenes. In the recent years, mechanism, a paramagnetic CO-inhibited species can be trapped by direct evidence for the cis-dihydroxylation of N-methyl-2-pyridone incubation of hydrogenase under a controlled CO atmosphere. The by NDOS has been reported in our laboratory 1,2; a major resulting Ni-CO species displays light-sensitivity properties, as dihydroxylated metabolite, N-methyl-cis-5,6-dihydro-5,6- observed for the Ni-C species (2). Upon irradiation monitored by dihydroxy-2-pyridone was obtained from this substrate. EPR, we carefully explored the photosensitivity of the Ni-CO species and subsequent thermal recombination processes. This relates to the photodissociation of the Ni-bound CO and the concomitant proton transfer between terminal cysteine ligands of the Ni ion. This strategy has been used with hydrogenase mutants for which The study presented here was initiated to extend this observation by intramolecular gas diffusion was hampered by mutating residues in investigating the effect of new substituents on 2-pyridone and 2- the channel close to the active site. quinolone rings in the NDO-catalyzed reaction. The potential (1) Montet, Y.; Amara, P.; Volbeda, A.; Vernede, X.; Hatchikian, asymmetric dihydroxylated derivatives may be used in the further E.C; Field, M.J.; Frey, M.; Fontecilla-Camps, J.C. NPG, 1997, 4, synthetic elaboration3 of a number of biologically important 523-526. compounds, such as unnatural amino acids, azasugars, alkaloids, (2) Dole, F., Fournel, A., Magro, V., Hatchikian, E.C., Bertrand, P., etc. Guigliarelli, B., Biochemistry, 1997, 36, 7847-7854

123 S74 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

The major products obtained were fully characterized and revealed pH, the associated changes in redox potentials, as well as the that for several substrates NDOS have catalyzed also extra-cyclic reactivity towards external ligands (NO) were complementary hydroxylations and N-dealkylation. studied by the conjunction of UV-visible, CD, NMR and EPR spectroscopies and electrochemistry. Since the square wave voltammograms obtained correspond to four successive one- electron processes, the experimental data were fitted to the theoretical predictions for four similar but non-equivalent redox sites. The binding of NO to the high-spin forms of D.vulgaris Hildenborough cytochrome c3 indicates that low-spin/high-spin equilibrium may enhance the reactivity of the hemes towards the binding and eventually catalysis of small molecules [3]. [1] Coletta, M., Catarino, T., LeGall, J. and Xavier, A.V., References: Eur.J.Biochem. 1991, 202, 1101-1106. 1- Modyanova, L.et al., Tetrahedron Lett. 2000, 41, 3865 [2] Rydberg, P., Sigfridsson, E. and Ryde, U., 2- Boyd, D.R. et al., Can. J. Chem. 2002, 80, 589 J.Biol.Inorg.Chem.,2004, 9, 203-223 3- Endoma, M.A. et al, Org. Proc. Res. Dev., 2002, 6, 525 [3] Santos, M., Santos, M.M.C., Gonçalves, M.L.S., Costa, C., Romão, J.C., Moura, J.J.G; Redox chemistry of low-pH forms of tetrahemic cytochrome c3 , J.Inorg.Biochem., 2006, 100, 2009- P116 2016. Magnetic circular dichroism of dicobalt model complexes Acknowledgments: This work is within the research project of metallohydrolases POCI/QUI/55743/2004. Seung-An Chyun, James A. Larrabee, Middlebury College, Middlebury, VT, USA. Contact e-mail: [email protected] P118 Radiolytic cryoreduction and cryooxidation of ferrous The ȝ-hyroxo-ȝ-carboxylato-dimetal core is a common structural hemoproteins: EPR and ENDOR evidence for feature in metallohydrolase active sites such as aminopeptidase and conformational substates of pentacoordinate ferrous glycerophosphodiesterase. An interesting feature of the Co(II)- substituted enzymes is the extent of magnetic exchange coupling hemoproteins Roman Davydov, Brian M. Hoffman, Northwestern University, between two high-spin Co(II) ions. Magnetic circular dichroism Evanston, IL, USA. (MCD) can be used to measure the magnitude of exchange coupling Contact e-mail: [email protected] between Co(II) ions. To demonstrate the principle, a study of two II II isostructural dicobalt complexes, [Co Co (ȝ-O2CCH3)2(ȝ- II III Ȗ-Irradiation of frozen solutions of ferrous hemoproteins at 77K OH)L ]PF (1) and [Co Co (ȝ-O CCH ) (ȝ-OH)L ](PF ) (2) (L = 2 6 2 3 2 2 6 2 generates EPR active singly-reduced low-valent hemes and Nƍ, NƎ, NƎƍ-triazacyclononane), was conducted. In 1 both Co(II) oxidized iron(III) heme centers, each trapped in the nonequilibrium ions are high-spin while in 2 the Co(III) is diamagnetic; there is no conformation of its ferrous precursor. The one-electron reduction possibility of coupling in 2. The low temperature MCD spectra of 1 products of pentacoordinate (S=2) ferrous , peroxidases, and and 2 are nearly identical having peaks at 483, 506 and 535 nm cytochrome P450cam show EPR and ENDOR spectra characteristic which arise from the 4T ĺ4T (P) d-d transition in Co(II). The 1g 1g of (3d7) Fe(I) species. Cryogenerated low-valent deoxyhemoglobin MCD spectrum of 1 is twice as intense as that of 2 because and deoxymyoglobin also exhibit an S=3/2 spin state comprised of a diamagnetic Co(III) is MCD silent. The magnetization plots for 1 pentacoordinate (S=2) Fe(II) ion antiferromagnetically coupled to a and 2 are very different, indicating that the ground states are porphyrin -anion radical. The EPR spectra of cryoreduced different. Spin-orbit coupling and low symmetry combine to create ʌ deoxyhemoglobin and deoxymyoglobin reveal three a pseudo-Kramer’s doublet ground state in single-ion 6-coordinate spectroscopically distinct low-valent species, which indicates that Co(II), and the magnetization data from 2 fit this model very well the deoxy precursors adopt different conformational substates. with g= 4.8. In coupled binuclear Co(II), spin-orbit coupling and Reduction products of hexacoordinate ferrous cytochrome c and b low symmetry lead to a ground state manifold of two singlets and a 5 as well as those of Fe(II)-CO globins are Fe(II) porphyrin ʌ-anion doublet, separated by energies, E and E . The doublet is lower in 1 2 radical species. The data thus show that the presence of the 6-th energy for ferromagnetic coupling. The magnetization data for 1 fit axial ligand in the low-valent intermediates favors localization of an well to the ferromagnetically coupled model with a g=9.6, E =4.2 1 unpaired electron in the porphyrin e * orbitals, consistent with cm-1 and E =8.5 cm-1. g 2 previous results for low-valent iron porphyrins models. The cryogenerated low-valent hemoprotein intermediates relax to the P117 ferrous states at T > 200K. The spectroscopic properties of cryogenerated ferric cytochrome C and b5 are similar to these for Redox chemistry of low-pH forms of tetrahemic the resting ferric states. The products of cryooxidation of cytochrome c3 pentacoordinate ferrous globins and peroxidases as well as of the 1 1 Margarida M. Correia dos Santos , Mara Santos , Maria de low-spin complexes of the ferrous globins with CO show EPR Lurdes Simões Gonçalves1, Cristina Costa2, José J. G. Moura2, eff spectra with gӆ < 6 characteristic of the quantum-mechanically Jorge C. Romão3, 1Centro de Química Estrutural,IST, Lisboa, 2 admixed S=3/2, 5/2 ground states. The trapped species relax to the Portugal; REQUIMTE-CQFB, FCT-UNL, Caparica, Portugal; equilibrium ferric states upon annealing at T > 190K. 3Centro de Física Teórica de Partículas, IST, Lisboa, Portugal. Contact e-mail: [email protected]

Desulfovibrio vulgaris Hildenborough cytochrome c3 contains four hemes in a low-spin state with bis-histidinyl coordination. High- spin forms of cytochrome c3 can be generated by protonation of the axial ligands [1,2]. In this work, high-spin forms of cytochrome c3 were generated by protonation of the axial histidine ligands in order to probe spin equilibrium (high/low), and to generate putative reactive species towards external ligands. The spin alterations occurring at extreme 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S75

P119 P121

Electron transfer complex between cytochrome c552 and Copper resistance proteins PcoA and PcoC from N2O reductase (N2OR) from Pseudomonas nautica Escherichia coli: expression, characterisation and Simone Dell'Acqua, Sofia R. Pauleta, Ludwig Krippahl, Alice S. interaction study Pereira, Isabel Moura, José J. G. Moura, REQUIMTE/CQFB, Karrera Y. Djoko, Zhiguang Xiao, Anthony G. Wedd, School of Departamento de Química, Faculdade de Ciências e Tecnologia, Chemistry and Bio21 Institute, University of Melbourne, Parkville, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. VIC, Australia. Contact e-mail: [email protected] Contact e-mail: [email protected]

The enzyme N2O reductase (N2OR) catalyses the final step of Copper is a micronutrient required for correct cellular functions but denitrification, the two electron reduction of N2O to N2. The is toxic in excess. However, a strain of E. coli isolated from the recently solved structures of N2OR from Pseudomonas nautica (Pn) faeces of copper-fed pigs can survive in copper-rich environments and Paracoccus denitrificans revealed the presence of a functional (up to 10 mM CuSO4). This resistance is attributed to a plasmid- homodimer containing two different multicopper sites: CuA and borne pco operon. High copper levels induce expression of the CuZ. CuA is a binuclear copper site that transfers electrons to the operon proteins PcoABCDERS that interact in the periplasm to catalytic CuZ site, a novel mixed valence compound with a sulfide confer copper resistance. ion bridging a distorted tetrahedron of copper atoms. In order to Both PcoA and PcoC are soluble proteins. We have demonstrated clarify the complex mechanism of activation and catalysis of this recently that PcoC (10.5 kDa) features two separate binding sites -13 enzyme we studied the interaction with the probable physiological (Kd ~ 10 ) specific for Cu(I) and Cu(II) and effects intermolecular electron donor: cytochrome c552 from Pn. Activity tests show that transfers of bound Cu(I) and Cu(II) with or without a change in the reduced cyt c552 is able to transfer electrons to CuA promoting oxidation state. Apo- and Cu(I)Cu(II)-forms are intermediates in the 1 the catalysis of N2O. The nature of the short-lived ET complex is two-step redox transfer processes. investigated using docking analysis performed with BiGGER1. This We have also expressed and isolated PcoA (63.7 Da). Preliminary study shows a strong affinity of this cytochrome to a hydrophobic data suggest that it is a laccase-type multicopper oxidase that binds patch located near CuA centre and putative complexes involving a multiple copper ions, including a T1 blue copper and a trinuclear small group of key residues are proposed. The recognition of the copper cluster at the active site.2 It catalyses the oxidation of two partners is driven by electrostatic interaction, while common laccase substrates, including para-phenylenediamine and hydrophobic contacts produce the fine orientation at short distances. 2,6-dimethoxyphenol, and requires excess copper for activity. In comparison, horse heart cytochrome c isn’t able to transfer Interactions between PcoA and PcoC under various conditions electrons to N2OR from Pn and the docking analysis of this non- provide insight into how these two members of the pco operon physiological electrostatic ET complex doesn’t point to a specific proteins function cooperatively in the periplasm and contribute to interaction. Kinetic studies and protein-protein 1H-NMR titrations this copper resistance in E. coli. Details of the expression, isolation are in progress to completely characterize this ET complex. and interaction study will be provided and discussed. 1.PalmaPN et al.,(2000) Proteins 39, 372 1. Djoko, K. Y.; Xiao, Z.; Wedd, A. G. Inorg. Chem. 2007, In press. S.D. is supported by a FCT-PhD grant (SFRH/BD/30414/2006) 2. Rensing, C.; Grass, G. FEMS Microbiol. Rev. 2003, 27, 197-213.

P120 P122 Study on the structure-property-reactivity-function Stability, reactivity, and mechanism of Alkane relationship of human neuronal growth inhibitory factor monooxygenase (AlkB) and its interaction with amyloid beta peptide Andrew Drabek1, Kate Luddy1, Rachel N. Austin1, John T. Zhi-Chun Ding, Zhong-Xian Huang, Chemistry Department, Groves2, Dayi Deng2, Jan van Beilen3, 1Bates College, Lewiston, Shanghai, China. ME, USA; 2Princeton University, Princeton, NJ, USA; 3ETH Contact e-mail: [email protected] Hönggerberg, Zurich, Switzerland. Contact e-mail: [email protected] Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), can inhibit the outgrowth of Alkane monooxygenase (AlkB) is a membrane-spanning diiron neuronal cells in the presence of brain extracts, a property not alkane hydoxylase that is widely contained in bacteria that catalyze shared by MT-1/-2. the oxidation of alkanes in the environment. Single mutations in a In order to study the structure-property-reactivity-function residue located in the presumed membrane-spanning substrate relationship, including the roles played by the particular residues channel alter the substrate specificity of alkane monooxygenase and by particular structural elements and to illustrate the possible (AlkB) and point to a key role for a substrate channel. Difficulties molecular mechanism of the bioactivity of hGIF a systematic site- maintaining activity through purification have limited studies on direct mutagenesis on hGIF has been done. The structure of ȕ- this important metalloenzyme. Results presented include procedures domain of hGIF, which was mainly responsible for the bioactivity for generating and characterizing stable cell free extracts from of the entire protein, was predicted by the molecular dynamics several different sources of AlkB, including those with mutations simulation for the first time. The properties of hGIF and its mutants that alter substrate specificity. These stable cell free extracts were characterized by a series of spectroscopy, the reactions of enabled characterization of reaction mechanisms using diagnostic hGIF and its variants with DTNB, EDTA and SNOC were substrates. Three diagnostic substrates - norcarane, bicyclohexane, examined, and finally the bioactivity of hGIF variants were assayed and bicyclopentane - were used. These substrates differ in size and by neuron culture experiments. rearrangement rate, facilitating a thorough characterization of From these studies we conclude that the bioactivity of hGIF is reaction mechanism. Evidence for a long-lived substrate radical is regulated by various factors, including the TCPCP motif, domain- very clear in all three substrates. domain interactions, the structure and stability of the metal-thiolate Acknowledgements: Financial support from the NSF through the cluster and other unknown factors. Environmental Molecular Science Institute, CEBIC (Center for The interaction between hGIF and amyloid beta peptide (Aȕ) is Environmental Bioinorganic Chemistry at Princeton University) another part of our research. It was reported that the level of hGIF CHE-0221978 (RNA, and JTG), CHE-0316301 (JTG), MCB- mRNA was down-regulated in AD brains and hGIF could inhibit 0078465 and CHE-0116233 (RNA), the Camille and Henry Dreyfus the aggregation of Aȕ in vitro. However, no detailed mechanism Foundation (RNA) and the National Institutes of Health, GM-32698 analysis has been carried out till now. The aim of our research is to (JTG) and GM072506 (RNA) is gratefully acknowledged. We also explore the mechanism how hGIF inhibits the aggregation of Aȕ thank Dr. John Eng and Dr. Dorothy Little for expert technical and provide information for potential drugs design for AD therapy. 123 S76 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

assistance with mass spectrometry and Charlotte Lehmann for - penetration into MPO when enzyme is bound to LDL. technical assistance with cell culturing and experiments. These compounds have been designed following a molecular docking analysis and included diacids (4), 4-substituted- and 5- substituted derivatives (5 and 6). Evaluation of these molecules P123 shows that MPO inhibition is not improved. Compounds 2 and 6 (X Spectroscopic and Mutagenesis studies on the Iron Sulfur = Br) have a slightly higher inhibitory effect. However, all the new Centers from the Fuscoredoxin of Desulfovibrio synthesized compounds and, particularly those with an additional desulfuricans acidic group, are weaker inhibitors of LDL oxidation. This could 1 1 2 1 originate from electrostatic repulsion between the acid groups of the Rui O. Duarte , Ana R. Ramos , Sara Costa , Alice S. Pereira , 1 1 1 ligands and negatively charged LDLs or from steric hindrance. Pedro Tavares , Isabel Moura , Requimte/CQFB/FCT, Almada, Portugal; 2ESTSL/IPL, Lisboa, Portugal. Contact e-mail: [email protected]

Fuscoredoxin is an unusual Fe-S protein from D. desulfuricans ATCC 27774. This protein contains two types of Fe-S clusters: a [4Fe-4S] cubane cluster and a novel one, [4Fe-2S-2O], which can attain four redox states. EPR and Mössbauer data reported some of the magnetic and redox properties, but they are still far from being understood. We cloned the gene in an expression vector (pGEX-6P- 1) and performed site-directed mutagenesis aiming to correlate the biochemical properties of the aminoacid involved in the metal center coordination with the spectroscopic data obtained for the protein, in particular the data on the iron related the atypical center. P125 We also have made a mutant consisting in the deletion of the first Structural, biochemical and functional characterizations 25 amino acids residues (from the N-terminal) to result in the of PerR specific loss of the [4Fe-4S], simplifying the data analysis. EPR and Abdelnasser El Ghazouani, iRTSV/ LCBM, Grenoble, France. Mössbauer studies are in progress to clarify the redox properties of Contact e-mail: [email protected] this mutant. These studies will also be an important tool for revealing the biological function of fuscoredoxin. Oxidative stress is generated by exposure to elevated levels of S. Macedo, E. Mitchell, C. Romão, S. Cooper, R. Coelho, M. Liu, Reactive Oxygen Species (ROS), such as hydrogen peroxide. To A. Xavier, J. LeGall, S. Bailey, C. Garner, W. Hagen, M. Teixeira, avoid or counter the harmful effects of ROS, cells constitutively M. Carrondo, P. Lindley, J. Biol. Inorg. Chem. 2002, 7, 514-525. express proteins to protect themselves and repair the damages. P. Tavares, A. Pereira, C. Krebs, N. Ravi, J. Moura, I. Moura, B. These proteins are under negative control of specific regulators Huynh, Biochemistry 1998, 37, 2830-2842. which are the central pieces of the inducible response to oxidative Acknowledgment: ROD thanks to the Fundação para a Ciência e stress present in cells. In Bacillus subtilis the zinc protein PerR, a Tecnologia and Requimte. peroxide sensor that binds DNA in the presence of a regulatory 2+ 2+ metal Mn or Fe , mediates the adaptive response to H2O2. It has been shown that Fe2+ or Mn2+ is essential for DNA binding and that P124 inactivation of PerR-Zn-Fe in the presence of H2O2 proceeds via the Conception of myeloperoxidase inhibitors based on metal catalyzed oxidation of at least 2 histidine residues that are flufenamic acid structure supposed to coordinate the Fe2+ ion. Pierre Van Antwerpen1, Martine Prevost2, François M. U. The present study reports the crystal structure of the PerR-Zn Dufrasne1, Patrick Moreau1, Karim Zouaoui Boudjeltia3, Thierry protein without its regulatory metal. Molecular modelling of the Franck4,5, Stephan Kohnen4,5, Sajida Babar6, Nicole Moguilevsky7, PerR-Zn structure led us to propose a potential binding site for the Michel Vanhaeverbeek3, Jean Ducobu8, Didier Serteyn4,5, Jean regulatory metal. The structural model for the active PerR protein Nève1, 1Laboratory of Organic Pharmaceutical Chemistry, Brussels, suggests a conformational change of the protein upon metallation. Belgium; 2Genomic and Structural Bioinformatic, Faculty of Taking advantage of the PerR-Zn structure, several mutations were Applied Sciences, Université Libre de Bruxelles, Brussels, carried out in order to introduce tryptophan residues as fluorescent Belgium; 3Laboratory of Experimental Medicine, CHU-Vesale, probes to monitor the dynamics of the protein. The results obtained Montigny-le-Tilleul, Belgium; 4Department of Clinical Sciences, with the PerR-Y77W mutant are described. Equine Clinic, Université de Liège, Liège, Belgium; 5Center for Recent work on the PerR-DNA interaction, in order to identify the Oxygen, Research and Development, Université de Liège, Liège, components of the highly specific recognition is also presented in Belgium; 6Laboratory of Applied Genetics, IBMM, Université light of protein-DNA cross-linking experiments and site-directed Libre de Bruxelles, Gosselies, Belgium; 7Technology Transfer mutagenesis studies. Office, FUNDP, Namur, Belgium; 8Department of Internal Medicine, CHU-Tivoli, La Louvière, Belgium. Contact e-mail: [email protected] P126 Zinc horse heart cytochrome c unfolding monitored by Myeloperoxidase (EC 1.11.1.7, MPO) is involved in several fluorescence resonance energy transfer pathologies, especially in atherosclerosis through its binding to Amy A. Ensign, Iris Jo, Todd Krauss, Kara L. Bren, University of LDL and lipoproteins oxidation. Attention has been focused on the Rochester, Rochester, NY, USA. development of MPO inhibitors with no success so far. Flufenamic Contact e-mail: [email protected] acid (1) has been reported as an in vitro MPO inhibitor. As a substrate of MPO compound I, it prevents the formation of HOCl - The physical basis for protein folding is one of the greatest from the MPO/H2O2/Cl system. It is metabolized into 5-chloro- and unknowns in science.1 Fluorescence resonance energy transfer 5-hydroxyflufenamic acids (2 and 3). Molecular docking predicts (FRET), when used as a “spectroscopic ruler”, is a method that that (1) interacts with the enzyme through aromatic stacking with gives information about intermolecular distances and is an attractive the heme and electrostatic bonds with binding site residues. A series method for the study of protein folding. However, FRET generally of inhibitor candidates were synthesized, based on the structure of requires the specific attachment of at least two dyes (a donor and an (1) with these expected characteristics: acceptor) to the protein; in practice, producing pure samples with - no or little HOCl scavenging, two dyes attached to specific sites has proved to be challenging.2 - enhanced interaction with the heme and/or binding site residues, 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S77

In order to eliminate the need to attach two dyes to perform FRET monochlorodimedone, with ClO2 being much more effective than experiments, we have chosen to study protein folding using horse hypochlorite. Enzymes of both peroxidase superfamilies cyt c in which the heme iron is replaced with Zn2+ (Zn-cyt c). (myeloperoxidase, horseradish peroxidase and catalase-peroxidase) Significantly, zinc porphyrin is an intrinsic fluorophore which can have been investigated in their reaction with chlorite by using the act as an acceptor of energy from a dye attached to the protein sequential-mixing stopped-flow technique. Bimolecular rate surface in FRET studies, eliminating the need to doubly label the constants at various pH values and a general reaction scheme are protein and minimizing perturbation to the system. The present presented and discussed with respect to known reduction potentials study examines folding probed by dyes attached to six different of enzyme intermediates (compound I/ferric peroxidase, compound sites on Zn-cyt c. This study confirms that the folding of horse cyt c I/compound II, compound II/ferric peroxidase) and of the couples - - - deviates from a two state folding model while making use of a ClO2/ClO2 and ClO2 /ClO , H2O. In addition, chlorine dioxide is novel fluorophore pair. shown to mediate the oxidation of ferric peroxidase to compound I 1. Dobson, C. M. Trends Biochem. Sci 1999, 24, 329-33. as well as inactivation of heme peroxidases. 2. Royer, C. A. Chem. Rev. 2006, 106, 1769-1784. *Shahangian, S., Hager, L.P. (1981) The Reaction of chloroperoxidase with chlorite and chlorine dioxide. J. Biol. Chem. 256, 6034-6040. P127 Kinetic studies of metal incorporation in Desulfovibrio gigas rubredoxin P129 Filipe Folgosa, Joana A. Santos, Alice S. Pereira, Isabel Moura, Redox states of Ferredoxin II from Desulfovibrio gigas José J. G. Moura, Pedro Tavares, REQIMTE, Faculdade de Raquel E. Grazina, Alice S. Pereira, Isabel Moura, José J. G. Ciências e Tecnologia, Caparica, Portugal. Moura, REQUIMTE, CQFB, Faculdade de Ciências e Tecnologia, Contact e-mail: [email protected] Universidade Nova de Lisboa, Caparica, Portugal. Contact e-mail: [email protected] Rubredoxin (Rd) is a small iron containing protein (~5kDa), present in several organisms [1]. This protein is often linked to electron Ferredoxins are simple iron-sulphur proteins that contain prosthetic transfer in biologic systems, working as an electron donor to other groups composed of iron and sulphur atoms and they play a proteins in many different pathways, like cellular detoxification for functional role in electron transfer processes relevant for sulphate- example [1, 2]. Electron transfer studies between rubredoxin and reducing bacteria (SRB) metabolism. Four distinct types of superoxide reductases (SOR) were recently published [3].To be able ferredoxins are found in SRB, containing [3Fe-4S], [4Fe-4S], [3Fe- to accomplish these functions the presence of the iron atom is 4S] plus [4Fe-4S] and 2×[4Fe-4S] clusters. Desulfovibrio gigas fundamental. ferredoxin II (DgFdII) is a small tetrameric protein of 58 amino However, it is possible to reconstitute Rd with different metals [4]. acids, which contains a single [3Fe-4S] cluster and a redox active Our present work is focused in the mechanism of metal internal disulfide bridge. DgFdII gene was cloned using the incorporation. polymerase chain reaction (PCR), inserted in the expression vector Several metals, like cobalt, zinc and iron, were used in the pET-21c and over expressed in Escherichia coli (E.coli). reconstitution of Desulfovibrio gigas Rd. All the studies were Electrochemical tools can provide important information for the preformed with overexpressed and purified protein. understanding of the redox and mechanistic/structural role of Fe-S clusters and to the interconversion process occurring between 3 Fe and 4 Fe clusters, as well as to the effect of the addition of an extra References: metal to form heterometal clusters of the type [M3Fe-4S]. 1 - Lovenberg W, Sobel BE. (1965), Proc. Natl. Acad. Sci. U. S. A., Characterization of the recombinant protein will be presented. 54:193 Direct electrochemistry (cyclic voltammetry and differential pulse 2 - Peterson JA, Kusunose M, Kusunose E, Coon MJ. (1967), J. voltammetry) will be used in order to detect all the redox transitions Biol. Chem., 242:4334 attainable by the core and to probe cluster interconversion. EPR will 3 - Rodrigues JV, Abreu IA, Cabelli D, Teixeira M. (2006), be used in complementary way. Biochemistry, 45: 9266 Acknowledgments: We would like to acknowledge to FCT-MCTES 4 - Moura I, Teixeira M, LeGall J, Moura JJ. (1991), J. Inorg. for financial support. Biochem., 44: 127 Moura, JJG et al., Methods in Enzymology, Vol. 243, 166-188 Acknowledgments: (1994), Peck, HD and LeGall, J, Ed., Acad Press. FF wishes to acknowledge to Fundação para a Ciência e Tecnologia Moura, JJG et al, 1994, Ferredoxins, in Methods in Enzymology, (FCT) for the grant SFRH/BD/18905/2004. J.H.D. Peck and J. LeGall Editors. Moreno, C et al., 1994, J. Inorg. Biochem., 53, 219-234.

P128 Chlorite as oxidant and reductant of heme peroxidases: a P130 mechanistic study Functional studies on a bacterioferritin from the Paul G. Furtmüller1, Christa Jakopitsch1, Holger Spalteholz2, anaerobe Desulfovibrio vulgaris Jürgen Arnhold2, Christian Obinger1, 1Department of Chemistry, Márcia Guilherme, Cristina G. Timóteo, Pedro Tavares, Alice S. Division of Biochemistry, BOKU – University of Natural Pereira, REQUIMTE, CQFB, Dep. de Química, Faculdade de Resources and Applied Life Sciences, Vienna, Austria; 2Institute of Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Medical Physics and Biophysics, School of Medicine, University of Portugal. Leipzig, Leipzig, Germany. Contact e-mail: [email protected] Contact e-mail: [email protected] Iron is an important nutrient, required in almost every aspects of - Heme peroxidases can use chlorite, ClO2 , in the absence of cellular function. However, at physiological pH and under oxidizing hydrogen peroxide to catalyze chlorination reactions.* We show conditions, the predominant form is Fe3+, which is highly insoluble. 2+ that chlorite acts both as oxidant (mediating the oxidation of ferric In the presence of O2, free Fe ions are extremely toxic. Ferritins enzymes to compound I) as well as one electron reductant are a broad superfamily of iron storage proteins present in all type (mediating the transformation compound I -> compound II -> ferric of organisms. Ferritin (Ft) and bacterioferritin (Bfr) are unique in peroxidase) and that all rates of these reactions increase with the sense that they perform dual functions of iron detoxification, by decreasing pH. The corresponding reaction products, hypochlorite oxidizing the Fe2+ ions in solution and iron sequestration by storing - 3+ (ClO ) and chlorine dioxide (ClO2), mediate the chlorination of the oxidized Fe ions in its inner protein cavity in the form of 123 S78 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

ferrihydrate mineral (1,2). They are large spherical proteins University, Chicago, IL, USA. composed of 24 subunits with a nanocavity of about 80 Å. Bfrs are Contact e-mail: [email protected] heme containing proteins with a b-type heme per dimer. A Bfr encoding gene was isolated from the Desulfovibrio vulgaris genome Particulate methane monooxygenase (pMMO) catalyzes the and overexpressed in Escherichia coli. Biochemical, kinetic and conversion of methane to methanol in methanotrophic bacteria. The spectroscopic studies on the recombinant D. vulgaris Bfr and a crystal structure of pMMO from Methylococcus capsulatus (Bath) variant (E59A) will be presented. revealed three metal centers: a dinuclear copper center and a [1] Liu, X. and Theil, E.C., Acc. Chem. Ber 2005, 38, 167. mononuclear copper center in the soluble portion of the protein and [2] Carrondo, M.A., EMBO J. 2003, 22, 1959. a zinc center in the transmembrane region.1 The zinc in the Aknowledgment: M.G., C.G.T. and A.S.P. wishes to acknowledge transmembrane site was derived from the crystallization buffer, and to Fundação para a Ciência e Tecnologia for financial support, the physiological occupant of this site was not identified. To SFRH/BD/18825/2004, SFRH/BPD/14863/2003 and address this issue and other questions left unanswered by the M. POCTI/QUI/47273/02, respectively. capsulatus (Bath) pMMO structure, we have characterized pMMO from a related organism, Methylosinus trichosporium OB3b. pMMO from M. trichosporium OB3b has been crystallized and a P131 low resolution structure determined. The structure is similar to that Magnetic circular dichroism study of a cobalt derivative of M. capsulatus (Bath) pMMO. Anomalous Fourier maps of Enterobacter aerogenes GpdQ calculated using data collected near the Cu, Zn and Fe absorption Kieran S. Hadler1, Mark J. Riley1, Lawrence R. Gahan1, Gerhard edges provide new insight into the identities and functional Schenk1, James A. Larrabee2, 1The University of Queensland, St relevance of the metal ions. According to Cu XAS data, the Lucia, Australia; 2Middlebury College, Middlebury, VT, USA. dinuclear copper center found in M. capsulatus (Bath) pMMO is Contact e-mail: [email protected] also present in M. trichosporium OB3b pMMO. The EPR spectrum of purified M. trichosporium OB3b is similar to that of M. The binuclear metalloenzyme glycerophosphodiesterase (GpdQ) is capsulatus (Bath) pMMO, with a typical type 2 signal and no evidence for a trinuclear cluster. the only enzyme known that is capable of catalysing the hydrolysis 1 of phosphoester bonds in mono-, di- and triesters. The protein binds Lieberman, R. L.; Rosenzweig, A. C. Nature 2005, 434, 177-182. one metal tightly and one loosely in the active site and thus activity is dependent on the concentration and identity of metals in the local environment. Magnetic circular dichroism (MCD) was used to P133 probe the binding and coordination of cobalt(II) in the active site. Delay time-resolved X-ray crystallographic study of Fe- The spectra reveal that addition of two equivalents of cobalt to the type nitrile hydratase apo-enzyme gives rise to only one protein MCD transition Kouichi Hashimoto, Masafumi Yohda, Masafumi Odaka, Faculty corresponding to a six-coordinate site. Addition of a further 48 of Technology, Tokyo University of Agriculture and Technology, equivalents leads to a second transition, corresponding to a partially Tokyo, Japan. occupied 5-coordinate site. The observed transitions and Contact e-mail: [email protected] coordination number were verified by angular overlap model (AOM) calculations. MCD of the phosphate inhibited species Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non- allows visualisation of the transition state involved in catalysis. heme iron enzyme having post-translationally modified cysteine Analysis of these spectra indicates that the coordination of each ligands, ĮCys112-SO2- and ĮCys114-SO-. NHase is widely used for metal is conserved, supporting the proposal that the phosphate industrial production of acrylamide, but its catalytic mechanism is displaces the bridging hydroxide during the phosphorolysis. In very limited. tert-butylisocyanide (t-BuNC) is a strong competitive addition, intensification of the 5-coordinate transition suggests that inhibitor with a Ki of 5 uM. Recently, we found that NHase the phosphate aids in assembly of the catalytic centre by anchoring catalyzes the conversion of t-BuNC to tert-butylamine. Since the the loosely bound metal in the second site. Magnetisation behaviour turnover rate for t-BuNC is much lower than that for nitriles, t- of the cobalt ions were studied and indicates an uncoupled system BuNC is likely to be a good substrate for studying the reaction with an isolated pseudo-Kramer doublet ground state in all species. mechanism of NHase. Here, we performed the time-resolved crystal structure analysis of NHase using t-BuNC as a substrate. t-BuNC was soaked to crystals of the nitrosylated inactive NHase. The catalytic reaction was started by photo-induced denitrosylation. At the reaction-elasped times of 0, 18, 70, 120, 340 and 440 mins, the reaction was stopped by flash-cooling of nitrogen gas and the X-ray diffraction data set were collected. At 0 min, a t-BuNC molecule was located in the reaction cavity but its -NC group was not interacted with NHase. At 70 and 120 mins, the carbon atom of the NC group in t-BuNC was coordinated to the iron at a distance of 2.07 Å. Interestingly, two electron densities maps were appeared at a distance of 1.26 Å from the carbon atom of the -NC group in the 440 min complex. The structure is likely to represent the enzyme- reaction intermediate complex. Based on the results obtained, the catalytic mechanism of NHase will be discussed.

P134 Peroxidase activity of hemoprotein reconstituted with iron porphycene P132 Takashi Hayashi, Takashi Matsuo, Department of Applied The metal centers of particulate methane monooxygenase Chemistry, Osaka University, Suita, Japan. from Methylosinus trichosporium OB3b Contact e-mail: [email protected] Amanda S. Hakemian1, Kalyan C. Kondapalli2, Joshua Telser1,3, Iron porphycene (FePc), a structural isomer of iron porphyrin, is an Brian M. Hoffman1, Timothy L. Stemmler2, Amy C. Rosenzweig1, attractive prosthetic group for hemoproteins. In our previous study, 1Northwestern University, Evanston, IL, USA; 2Wayne State we have reported that myoglobin reconstituted with FePc had University School of Medicine, Detroit, MI, USA; 3Roosevelt 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S79

extremely high O2 affinity, whereas unfavorable autoxidation was and a 40 % occupancy of the right-handed spiral conformation, remarkably slow due to the small rate constant of O2 dissociation. while molecule B has a disulfide bond only in the right-handed The interesting behavior may be due to the decrease of the spiral conformation. Furthermore, the A molecules create dimers macrocyclic ligand framework. Recently, we also focused on the through a ȕ-sheet interaction, while the B molecules are packed as peroxidase and peroxygenase activities of the hemoproteins monomers in a less rigid position. reconstituted with FePc. The H2O2-dependent oxidations of [1] M.S. Nielsen, P. Harris, B.L. Ooi and H.E.M. Christensen. guaiacol, thioanisole and styrene catalyzed by myoglobin Biochemistry 2004, 43, 5188-5194 reconstituted with FePc gave the corresponding oxidation and/or oxygenation products with ca. 10-fold acceleration compared to the native protein. In addition, the large amounts of H2O2 led to the conversion of compound II-like species of FePc into the compound III-like species, oxygenated FePc, in the myoglobin heme pocket. Furthermore, horseradish peroxidase (HRP) reconstituted with FePc was also prepared by the conventional method. The peroxygenase activity of the reconstituted protein toward thioanisole oxidation was larger than that of native HRP by 10-fold. The highly oxidative intermediates, compound I and II of FePc were successively detected by stopped-flow technique. In this presentation, we will discuss the reactivity of FePc in the hemoprotein pocket. T. Hayashi et al. Acc. Chem. Res. 2002, 35, 35-43. T. Hayashi et al. J. Am. Chem. Soc. 2004, 126, 16007-16017. T. Hayashi et al. Inorg. Chem. 2006, 45, 10530-10536. P137 P135 Alterations in the thermostability of apo and zinc-bound Identification of a heme axial ligand of heme-regulated wild type and ALS mutant CuZnSOD eukaryotic initiation factor 2 alpha kinase (HRI): Role of Matthew H. S. Clement, Edith B. Gralla, Lindsay E. Kane, Kevin the Cys-Pro motif in heme regulation W. Sea, Joan Selverstone Valentine, University of California, Los Jotaro Igarashi, Aya Iizuka, Motohiko Murase, Marketa Angeles, Los Angeles, CA, USA. Martinkova, Toru Shimizu, Institute of Multidisciplinary Research Contact e-mail: [email protected] for Advanced Materials, Tohoku University, Sendai, Japan. Contact e-mail: [email protected] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results in death within two to five years. The Heme-regulated eIF2Į kinase (heme-regulated inhibitor [HRI]) homodimeric antioxidant enzyme Cu-Zn superoxide dismutase plays a critical role in the regulation of protein synthesis by heme (SOD1) has been implicated in two percent of ALS cases. Over 100 iron. HRI senses and mediates responses to changes in the heme mutations leading to ALS have been documented in SOD1. Our concentration. However, little is known regarding how heme binds laboratory has characterized biophysically a number of these mutant to the catalytic domain and regulates catalysis, the stoichiometry of proteins and found that they fall into two general classes, pseudo- heme binding, and which residues are axial ligands for the heme wild type or metal-binding region mutants. We have found that iron. From our spectroscopic studies of an N-terminal truncated many of the pseudo-wild type ALS mutations destabilize the apo- mutant lacking the first 145 amino acids, it was suggested that one protein. Previous studies have indicated that major structural of the heme axial ligands should be a Cys residue located in the C- rearrangements occur upon the binding of one zinc to the wild type terminal catalytic domain. To determine which Cys residue is apo protein dimer. Here we extend our studies of the ALS mutant responsible for the heme binding, we generated site-directed proteins to include the thermal stability of the zinc bound forms mutants at six Cys residues. Heme binding abilities of the Cys using differential scanning calorimetry. Surprisingly, the mutant mutants were clearly different from that of the wild-type protein. proteins with one zinc bound have similar stability to wild type, The role of the Cys-Pro motif in the heme binding will be dissected. despite their differing (lower) stability in the apo form. With two Relationships between the heme binding and catalytic regulation zinc ions bound, the ALS mutant proteins once again exhibit a will also be discussed. wider range of thermal stability. Identifying causes of the differing (1) Igarashi, J., Sato, A., Kitagawa, T., Yoshimura, T., Yamauchi, thermal stabilities of the apo and metallated forms of the proteins S., Sagami, I., and Shimizu, T. (2004) J. Biol. Chem., 279, 15752. will help us better understand the nature of the toxicity of the (2) Miksanova, M., Igarashi, J., Minami, M., Sagami, I., Yamauchi, mutant proteins. S., Kurokawa, H., and Shimizu, T. (2006) Biochemistry, 45, 9894. P138 P136 Direct electrochemistry of amicyanin derived from The crystal structure of the all cysteinyl coordinated Paracoccus denitrificans immobilized on a electrode via a D14C mutant of [4Fe-4S] Pyrococcus furiosus ferredoxin phenylglyoxal derivative Monika N. Johannessen, Michael S. Nielsen, Pernille Harris, Bee Ayako Kaneko, Nobuhumi Nakamura, Hiroyuki Ohno, Tokyo L. Ooi, Hans E. M. Christensen, Department of Chemistry, University of Agriculture & Technology, Koganei, Tokyo, Japan. Technical University of Denmark, Kgs. Lyngby, Denmark. Contact e-mail: [email protected] Contact e-mail: [email protected] The oriented immobilization of redox proteins on an electrode is The structure of the all cysteinyl coordinated D14C mutant of [4Fe- expected to control a electron transfer reaction between the protein 4S] ferredoxin (Fd) from the hyperthermophilic archaeon and the electrode. There are methods for the oriented Pyrococcus furiosus has been determined to 1.7 Å resolution from a immobilization using specific amino acids within protein. A keto crystal belonging to space group C2221 with two molecules in the aldehyde group of phenylglyoxal selectively reacts with a guanidino asymmetric unit. The structure has been solved with molecular group of an arginine residue. In this study, amicyanin (Ami) from replacement using the [3Fe-4S] ferredoxin from P. furiosus [1] as a Paracoccus denitrificans have been immobilized on the electrode search model. The two molecules in the asymmetric unit are via Arg by using p-carboxyethylphenylglyoxal (CEPG). Ami is a different. The A molecule contains a double conformation of the blue copper protein which mediates electron transfer between disulfide bond which exists in a 60 % occupancy of the left-handed methylamine dehydrogenase and cytochrome c-551i. Furthermore, to 123 S80 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

investigate the effect of the orientation of Ami toward an electrode Homogeneous AKs with the ratio metal/protein equal 1/1 (where on its electrochemical properties, we constructed three Ami mutans metals were Co2+, Zn2+, Fe2+) were obtained. Apo-AK was obtained (R48A, R99A, R48A/R99A). in vivo conditions in inclusion bodies by following refolding in The gold electrode was immersed into 2-mercaptoethanol (ME), presence of detergent NDSB256 as well as in vitro conditions using and then, immersed in CEPG solution including NHS and EDC. phenylmercurihydroxyde as substituted agent and DTT and TCEP The modified electrodes were soaked in the protein solution. The as reducing agents. Metal chelated and apo-AK forms were redox responses using ME-Ami and ME/CEPG-Ami electrodes spectroscopically (UV/Visible, EPR) and kinetically characterized were observed by cyclic voltammetry. This result suggests that Ami and compared. For metal containing forms Vmax for ADP and adsorbed nonspecifically on the ME-modified electrode. Therefore, MgADP can be represented in following order Zn-AK > Co-AK = to remove the nonspecifically absorbed Ami, Ami-immobilized Fe-AK. In the same time KM values for MgADP for all metal electrodes were immersed in phosphate buffer containing 1 M chelated forms are the same but KM values for ADP are vary (Co- NaCl. As a result, the redox response using the ME-Ami electrode AK > Zn-AK > Fe-AK). The kinetic properties of apo-AK are was diminished. When the ME/CEPG-Ami electrode was used, the different with comparison to metal chelated forms. Thus specific redox response was still observed. The results suggest that CEPG is activity of it is only 20% from metal chelated forms.This work was a useful compound to immobilize protein via Arg on the electrode supported by POCI/QUI/59119/2004 from FCT and No E-62/06 surface. We will also report the electrochemistry using the Ami from CRUP, SFRH/BPD/28380/2006 and SFRH/BD/24744/2005. mutants immobilized on the electrode. P141 P139 High Resolution Pulsed EPR and 17O-NMR Studies of Environment of "proximal" heme pocket in myoglobin Molybdoenzyme Model Complexes multiple mutants as revealed by resonance Raman Eric L. Klein, Andrei V. Astashkin, John H. Enemark, University spectroscopy of Arizona, Tucson, AZ, USA. Kohei Kiyota, Yoshikazu Tomisugi, Tadayuki Uno, Graduate Contact e-mail: [email protected] School of Pharmaceutical Sciences, Osaka University, Suita, Japan. Contact e-mail: [email protected] Sulfite oxidase (SO) is a vital molybdoenzyme that catalyzes the 2- 2- oxidation of sulfite (SO3 ) to sulfate (SO4 ). A part of our effort to Porcine myoglobin (Mb) mutants having a ligand binding pocket in elucidate details of the catalytic mechanisms of molybdoenzymes, the heme proximal side was investigated by absorption and particularly that of SO, includes the study of well-defined model resonance Raman spectroscopic methods. complexes that can provide a means of interpreting specific Mb is an oxygen storage protein which has been the subject of spectroscopic features of enzymes and their proposed reaction intense research over the last few decades. The heme iron in wild- intermediates. The coordination of chloride (35,37Cl, I = 3/2) to type (WT) Mb is coordinated to the protein through the proximal Mo(V) in the low pH form of SO, for example, has been proposed His93, and exogenous small ligands including oxygen molecule based on indirect EPR and X-ray absorption evidence, and chloride bind to the distal iron coordination site. We focused on a quadruple coordination has also been invoked to explain some of the recurring mutant, H64V/V68H/H93A/H97F (VHAF), which uniquely has an features in the electron spin echo envelope modulation (ESEEM) endogenous distal His68 ligand and a binding pocket for an spectra of SO. However, the unambiguous identification of this exogenous ligand in the iron proximal side1). In order to make the ligand and a complete description of its interaction with Mo in the proximal pocket for exogenous ligands to access easily, we further SO active site have remained elusive. Here, the ESEEM of constructed a quadruple mutant, H64V/V68H/H93A/H97A oxomolybdenum(V) complexes that have chlorine nuclei in either (VHAA), in which the proximal Phe97 in VHAF is replaced by less the first or second coordination sphere will be presented. The bulky Ala. Stability of the bound heme, pH dependence, and ligand ESEEM of 17O (I = 5/2) labeled Mo(V) oxo/hydroxo complexes (CN-, CO) affinity to VHAA were studied using UV-vis absorption will also be presented, allowing benchmark hyperfine and nuclear technique, and the heme structure as well as its environment was quadrupole interaction parameters for Mo=17O and Mo-17OH investigated by resonance Raman spectroscopy. The VHAA mutant species to be determined and compared to similarly labeled SO. was revealed to be a six-coordinate high-spin species in the ferric Finally, using 17O-NMR, model studies relevant to the mechanism state and five-coordinate high-spin species in the ferrous state. The of oxygen atom transfer in the fully oxidized Mo(VI)O2 SO state Fe-CO stretching frequency of VHAA was higher than that of will be discussed. VHAF, suggesting more polar proximal environment similar to that in WT Mb. The pH dependence of VHAA, however, differed substantially from that of WT. Thus, VHAA was revealed to have a P142 unique binding pocket for exogenous ligands. A ferredoxin apo-state with molten globule 1) Uno T. et al., Biochemistry 42, 10191 (2003) characteristics: a role in iron-sulfur center assembly? Sónia S. Leal, Cláudio M. Gomes, Instituto de Tecnologia Química e Biologica, Oeiras, Portugal. P140 Contact e-mail: [email protected] Metal-chelated forms of adenylate kinase Anna V. Kladova, Sergey A. Bursakov, Olga Yu. Gavel, Isabel The biological insertion of Fe-S involves the interaction of Moura, Jose J. J. G. Moura, Universidade Nova de Lisboa, (metallo) chaperons with a partly folded target polypeptide. In this Caparica, Portugal. respect, the study of non-native protein conformations in iron-sulfur Contact e-mail: [email protected] proteins is relevant for the understanding of the folding process and cofactor assembly [1-6]. We have investigated the formation of a Adenylate kinases (AK) catalyze the reversible transfer of the molten globule state in a prokaryotic [3Fe4S][4Fe4S] ferredoxin terminal phosphate group from MgATP to AMP and contribute to which also contains a structural Zn2+centre [6]. the maintenance of constant level of cellular adenine nucleotides. Biophysical studies have allowed to dissect the sequence of thermal The adk gene from D. gigas was cloned and the protein unfolding events: Fe-S disassembly, loss of tertiary contacts and overproduced in E. coli BL21(DE3) strain using M63B1 minimal dissociation of the Zn2+ site which is simultaneous to alterations on medium. The method for metal containing recombinant AK the secondary structure. Upon cooling, an apo-ferredoxin state is production was developed for in vivo conditions, for the purpose to obtained, with characteristics of a molten globule: compactness understand the role of metals for activity and stability of AK. The identical to the native form; similar secondary structure evidenced optimal conditions (concentrations of metals and inductor IPTG, by far-UV CD; no near-UV CD detected tertiary contacts; and an time and temperature of incubation) were established. exposure of the hydrophobic surface [6]. 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S81

The possible physiological relevance of molten globule states in Fe- 2 I. Kawashima, T.D. Kennedy, M. Chino, B. Lane, 1992, Eur. J. S proteins and the hypothesis that their structural flexibility may be Biochem., 209, 971-976 important to the understanding of metal centre insertion are 3 C.A. Blindauer, M.D. Harrison, J.A. Parkinson, A.K. Robinson, discussed. J.S. Cavet, N.J. Robinson, P.J. Sadler, 2001, Proc. Natl. Acad. Sci. [1] Gomes, C.M., et al. J. Bio. Inorg. Chem. 1998, 3, 499-507 [2] USA, 98, 9593-9598 Moczygenba, C., et al. Protein Sci. 2001, 10, 1539-1548 [3] Leal, S.S. et al. J. Biol. Inorg. Chem. 2004, 9, 987-996 [4] Rocha, R., Leal S.S., et al. Biochemistry 2006, 45, 10376-10384 [5] Leal, S.S. P145 et al. Biol. Chem. 2005, 386, 1295-1300 [6] Leal, S.S. et al. Novel Nitric Oxide Dioxygenase, SHP (Sphaeroides Heme Proteins 2007 in press Protein), and New detoxification mechanism Bor-Ran Li1, J. L. Ross Anderson1, Caroline S. Miles2, Christopher G. Mowat1, Graeme A. Reid2, Stephen K. Chapman1, 1School of P143 Chemistry, University of Edinburgh, United Kingdom; 2School of Impact of the first coordination sphere of Fe2+ in Dke1 Biology, University of Edinburgh, United Kingdom. on cofactor affinity Contact e-mail: [email protected] Stefan Leitgeb, Grit Straganz, Bernd Nidetzky, Institute of Biotechnology and Biochemical Engineering, Graz University of SHP (Sphaeroides Heme Protein) is an oxygen-binding cytochrome, Technology, Graz, Austria. typically found in photosynthetic and metal-reducing bacteria. Like Contact e-mail: [email protected] other oxygen-binding cytochromes, it not only binds oxygen but also nitric oxide, with similar “on-rates”. SHP is found in 16 Diketone cleaving enzyme (Dke1) from Acinetobacter johnsonii is a different species of bacteria and, exceptionally, is the only known c- non-heme iron dependent dioxygenase that degrades its natural type cytochrome to have asparagine as a heme ligand. The 3- substrate acetylacetone by oxygenative C-C bond cleavage. The dimensional structure of SHP was solved in 2000 [1]. The redox metal binding site of this enzyme is similar to other dioxygenases partner for SHP is thought to be another cytochrome known as that show a cupin fold and consists of three histidine-residues. Since DHC (DiHeme Cytochrome c) [2]. In previous studies, we have the majority of analogous non-heme metal-dependent dioxygenases shown that DHC transfers electrons efficiently to SHP. DHC also possess a 2-His-1-carboxylate facial triad we performed binds tightly to SHP under low ionic strength conditions. However, mutagenesis experiments to replace the first-coordination sphere the biological function of SHP is not clear. An investigation of the histidine residues by carboxylate and carboxylamine residues to Integrated Microbial Genomes data base revealed that the genes for investigate the effects on activity and metal binding. Microdialysis SHP/DHC/Cyt b are side by side in a gene neighbourhood, and their equilibrium experiments combined with fluorescence titrations mRNAs are up-regulated when nitrite is the electron acceptor. showed the removal of a high affinity iron binding site in Dke1, Using biochemical methods, we have demonstrated that the consistent with the loss of diketone-cleaving activity. A second, oxyferrous form of SHP can react rapidly with nitric oxide to form low-affinity, iron binding site was identified that is not relevant for nitrate. Therefore, we propose that one function for SHP is as a catalysis. In contrast, the other divalent ions that were tested in nitric oxide dioxygenase. terms of binding - Cu2+, Ni2+, Zn2+, Mn2+ - only showed minor 1. Leys D, Backers K, Meyer TE, Hagen WR, Cusanovich MA, effects upon exchange of the first coordination sphere residues. We Van Beeumen JJ. J Biol Chem. 2000 May 26;275(21):16050-6. therefore found that by disrupting the 3-histidine coordination motif 2. Gibson HR, Mowat CG, Miles CS, Li BR, Leys D, Reid GA, in Dke1 we could selectively lower the enzymes affinity for Fe2+. Chapman SK. Biochemistry. 2006 May 23;45(20):6363-71.

P144 Novel cluster structure and histidine coordination in a plant metallothionein from wheat embryos Oksana I. Leszczyszyn, Claudia A. Blindauer, University of Warwick, Coventry, United Kingdom. Contact e-mail: [email protected]

Gene mining and expression studies have shown that plant metallothionein-like proteins display significant variation in sequence and biological function. However, information about their biochemical properties, metal-binding interactions and solution structure is considerably underrepresented in metallothionein research.1 We present a comprehensive characterisation of one plant MT exclusively expressed in the embryos of pre-germinating wheat 2 P146 seeds (EC). Unlike other metallothioneins, the CXC motifs of EC are arranged into three regions along the primary sequence, and as Metal binding properties of Heat-shock protein A (HspA) such the EC sequence cannot be aligned with those of mammalian Hongyan Li, Shujian Cun, Wei Xia, Ruiguang Ge, Marie C. M. origin. In addition, EC has two histidine residues, which are not Lin, Hongzhe Sun, Department of Chemistry, The University of present in mammalian metallothioneins, but have been shown to Hong Kong, Hong Kong, China. coordinate metal ions in bacterial metallothioneins.3 It is therefore Contact e-mail: [email protected] not surprising that the solution properties and structure differ from previously characterised metallothioneins. The Heat-Shock Protein A (HspA) is a member of GroES Using various multinuclear and multidimensional NMR chaperonin family, and it consists of 118 amino acids with a unique experiments, we demonstrate the involvement of histidine residues histidine/cystein-rich C-terminus, which is completely absent from in zinc ion coordination and their likely modes of binding, together most other GroES members. When being introduced into with analysis of backbone dynamics and support for a novel cluster Escherichia coli with urease gene cluster together, HspA has been [1] composition. 111Cd NMR and [1H,1H] TOCSY spectra reveal observed to be able to increase urease activity greatly , indicating interesting exchange behaviour in one of the clusters and also offer that the protein may play an important role for the process of nickel structural insights. transportation. Histidine-rich proteins are known for their abilities [2, 3] 1 O.I. Leszczyszyn, R. Schmid, C.A. Blindauer, 2007, Proteins: to bind to nickel although their biological functions are not well Struct., Func., Bioinf., Accepted manuscript understood. 123 S82 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

The complete coding sequence of hspA was cloned from the P148 genomic DNA of Helicobacter pylori (strain 26695) and the protein Elucidating the functional properties of metalloenzymes: was expressed in E. coli. and purified by nickel-affinity column and design and characterization of DF3 an artificial di-iron was verified by mass spectrometry. The aggregation behavior was protein studied via gel filtration chromatography. The metal binding 1 2 1 Ornella Maglio , Rafael Torres Martin de Rosales , Flavia Nastri , properties of both HspA and its C-termini histidine/cystein rich 1 1 3 Marina Faiella , Vincenzo Pavone , William F. DeGrado , Angela peptide were examined using UV-vis spectroscopy and the results 1 1 Lombardi , University of Naples "Federico II", Napoli, Italy; showed that HspA binds to nickel and bismuth with a stoichiometry 2 3 Imperial College London, London, United Kingdom; University of 2:1, similar to that of nickel binding to the peptide. Binding of of Pennsylvania, Philadelphia, PA, USA. both Ni2+ and Bi3+ to the protein is reversible and can be released Contact e-mail: [email protected] either by chelate ligands such as EDTA or by lowing pH. [1] Kansau I. et al. (1996) Mol Microbiol 22: 1013. A major objective in protein science is the design of novel enzymes [2] (a) Ge R. et al. (2006) Biochem. J. 393: 285 (b) Ge R. et al. with properties tailored to specific applications. This goal is (2006) J. Am. Chem. Soc. 128: 11330. becoming increasingly possible: the growing number of protein [3] Mehta N. et al. (2003) J. Bacteriol. 185: 726. sequences and known three-dimensional structures are providing essential information, which contribute to a deeper understanding of P147 the relationship between protein sequence, structure, and function. In this perspective, metalloprotein design provides a powerful Directed evolution of for improved activity approach, contributing to a more comprehensive understanding of towards chlorinated phenols the way metalloproteins function in biology, with the ultimate goal Timothy E. Machonkin1,2, Min-Sun Park2, Brian O'Mara2, Taylor of developing novel biocatalysts and sensing devices1. L. Weiss2, 1Whitman College, Walla Walla, WA, USA; 2University We approached the challenge of reproducing metalloprotein of Rochester, Rochester, NY, USA. activesites through the de novo protein-design. We centered our Contact e-mail: [email protected] attention on iron-containing proteins, and we developed the DF (Due Ferri) family of artificial proteins, as models of di-iron Tyrosinase holds great potential for the bioremediation of phenols proteins2. in wastewater, since it reacts with such compound to form A first subset of the DF family, DF1, consisted of two non- minimally soluble pigments that can be removed readily. It is covalently associated helix-loop-helix motifs, which bound the di- unique in being capable of catalyzing the hydroxylation and metal cofactor near the center of the structure. subsequent oxidation of phenols using only O2 and substrate DF1 analogues showed limited solubility in aqueous solution, without additional reducing equivalents or co-substrates. probably due to an inter-helical turn with strained conformation, We have developed an expression system for Streptomyces and an active site relatively inaccessible. To enhance the properties gluacescens tyrosinase that produces enzyme that is active in vivo in of this subset, we refined the structure by designing a new high yield, and have designed a strategy for the directed evolution symmetric helix-loop-helix dimer, DF3. DF3 contains changes into of tyrosinase mutants with improved activities towards non-native the sequence to improve metal ion access, and a new inter-helical phenol substrates. This approached yielded a series of mutants of loop. tyrosinase with improved activity towards chlorinated phenols, It shows improved solubility and active site accessibility, while specifically 4-chlorophenol. The first round of directed evolution retaining the unique native-like structure. yielded 25 mutants that were 50-150% more active towards 4- 1. Lu, Y. “Encyclopedia of Inorganic Chemistry”, 2nd-Ed. (King, chlorophenol. A second round of directed evolution yielded two B.-Ed.,Wiley) 2006, Vol. V, 3159. 2. Calhoun, J. R.; Nastri, F.; double-mutants with an approximately 5-fold increase in kcat Maglio, O.; Lombardi, A. & DeGrado, W. F. Biopolymers 2005, towards 4-chlorophenol. Detailed steady-state kinetic studies also 80, 264. showed modest improvements in kcat towards other chlorophenols, and decreased activity towards methylphenols. The mutations identified are mostly in the second-coordination sphere, and provide P149 an interesting basis for future studies of structure-function Role of the Threonine 101 residue on the stability of the correlations in this enzyme. heme active site of cytochrome P450cam: Multi- wavelength Circular Dichroism Studies Soumen K. Manna, Shyamalava Mazumdar, Tata Institute of Fundamental Research, Mumbai, India. Contact e-mail: [email protected]

The role of the Thr101 residue in the conformational property and the stability of the active site of cytochrome P450cam were studied using site-directed mutagenesis and multi-wavelength circular dichroism (CD) techniques. The T101V mutation did not change the substrate binding affinity of the enzyme but it was associated with subtle changes in the conformational anisotropy around the heme active site. Thermostability of the mutant and the wild type enzyme have been studied by temperature dependence of the far-UV CD as well as that of the visible CD bands that arise from the heme moiety along with differential scanning calorimetry. The analysis of the thermal unfolding data showed that the thermal stability of the secondary structure of the enzyme was unaffected by the mutation. But the mutation was found to decrease the thermostability of the active site in presence of the substrate. On the other hand, this mutation had no apparent effect on the thermostability of the substrate-free enzyme. These results suggested that the threonine residue stabilizes the heme centre in case of the substrate-bound species, possibly by hydrogen bonding with one of the heme- propionate side chains. Such hydrogen bonding of the heme- propionate with threonine is absent in the substrate-free enzyme. 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S83

This study also indicated that the multi-wavelength CD studies can verdoheme opening (Fig.). The reaction with ethyl hydroperoxide, help to identify the weak interactions in the active site of but not t-butyl and cumyl hydroperoxides, under reducing metalloenzymes. conditions afforded two linear tetrapyrroles without forming significant amount of biliverdin. The new products could also be prepared by chemical opening of verdoheme with sodium ethoxide, and were identified by HPLC and ESI-MS analysis as 1-ethoxy- and 19-ethoxy-deoxybiliverdins. The formation of the ethoxy- biliverdins unambiguously identifies the Fe-OOR species as a key intermediate in the EtOOH reaction, and strongly suggests the corresponding Fe-OOH mechanism for the physiologically relevant O2- and H2O2-dependent biliverdin production. This is similar to the well-established mechanism of the first oxygenation, meso- hydroxylation of heme. HO appears to be evolved to control the reactivity of the Fe-OOH complexes to allow addition of their terminal oxygen moieties to the porphyrin macrocycle.

P150 Redox states of Nitric Oxide Reductase from Pseudomonas nautica: Kinetic and Spectroscopic characterization Carlos E. Martins1, Alice S. Pereira1, Pedro Tavares1, Cristina M. Cordas1, Filipe Folgosa1, Cristina G. Timóteo1, Sunil Naik2, Boi Hanh Huynh2, José J. G. Moura1, Isabel Moura1, 1REQUIMTE/CQFB, Dept Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal; 2Department of Physics, Emory University, Atlanta, GA, USA. Contact e-mail: [email protected]

Nitric Oxide Reductase (NOR) from Pseudomonas (Ps.) nautica 617 is a membrane bound enzyme which belongs to the cNOR class. The as-isolated form is a heterodimer with a ~17 kDa (NORC) and a ~54 kDa (NORB) subunits. While NORC is a c-type monohemic subunit, NORB contains two b-type hemes and a non- heme iron atom. P152 The protein can be stabilized in three different redox forms: the as- Modulation of redox potential and activity via the isolated, ascorbate reduced and dithionite reduced, that exhibit peroxide shunt pathway by mutation of cytochrome P450 different spectroscopic and kinetic behavior. The application of an array of spectroscopic (UV/Visible, EPR and Mössbauer around the proximal heme ligand Hirotoshi Matsumura, Sayaka Omi, Masahiro Wakatabi, Akashi spectroscopies) in conjugation with visible stopped-flow and Ohtaki, Masafumi Yohda, Nobuhumi Nakamura, Hiroyuki Ohno, electrochemistry to the study of the different redox forms, allowed Department of Biotechnology and Life Science, Tokyo University the identification of the active form. Cytochrome c from the same 552 of Agriculture and Technology, Koganei, Tokyo, Japan. organism was, also, identified as the putative physiologic electron Contact e-mail: [email protected] donor in the nitric oxide reduction reaction catalyzed by NOR. Data will be shown on the bifunctionallity of Ps. nautica NOR, Cytochrome P450s are heme monooxygenases involved in a variety capable of NO reduction, as well as O reduction. 2 of oxidative metabolic reactions, which constitute a large heme- [1] Cordas, C.M., A.S. Pereira, C.E. Martins, C. G. Timoteo, I. thiolate protein family. The enzymes mainly catalyze the Moura, J.J.G. Moura, P. Tavares, Chembiochem., 2006, 7(12), monooxygenation of a variety of hydrophobic substrates. We 1878-81 reported overexpression and purification of cytochrome P450 from [2] Tavares, P., A.S. Pereira, J.J.G. Moura and I. Moura, J. Inorg. a thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7 Biochem., 2006, 100(12), 2087-100 (P450st), previously.1 It has been reported that the electronic [3] Girsch P. and de Vries S., Biochim. Biophys. Acta, 1997, 1318 environment around the heme group moiety and proximal heme (1-2), 202-16 ligand, Cys, affects the redox potential of cytochrome P450s.2 In Acknowledgements: CEM, CMC, FF, CGT wish to acknowledge to this study, we have constructed the P450st mutants substituted Fundação para a Ciência e Tecnologia for financial support: Phe310 to Ala and Ala320 to Gln, respectively. The double-point SFRH/BD/17840/2004; SFRH/BD/2917/2000; mutant changed at both F310A and A320Q has also been SFRH/BD/18905/2004; SFRH /BPD/14863/2003 respectively. constructed. Phe310 and Ala320 in P450st reside closely at the conserved cysteine ligand, Cys317 in P450st, which coordinates to P151 heme iron (Fig. 1). We have been investigated the effects of the substitutes on the electrochemical properties and thermostability. Ring opening mechanism of verdoheme catalyzed by The reduction potential and the catalytic activity with shunt heme oxygenase pathway of P450st and mutants have also been investigated. Toshitaka Matsui, Hiromichi Jin, Masao Ikeda-Saito, Tohoku 1. Oku, Y.; Ohtaki, A.; Kamitori, S.; Nakamura, N.; Yohda, M.; University, Sendai, Japan. Ohno, H.; Kawarabayasi, Y. J. Inorg. Biochem. 2004, 98, 1194- Contact e-mail: [email protected] 1199. 2. Ost, T.W.B.; Clark, J.; Mowat, C.G.; Miles, C.S.; Walkinshaw, Heme oxygenase (HO) catalyzes catabolism of heme to biliverdin, M.D.; Reid, G.A.; Chapman, S.K.; Daff, S. J. Am. Chem. Soc. 2003, CO and a free iron through three successive oxygenation steps. The 125, 15010-15020. third oxygenation, ring opening of verdoheme to yield biliverdin, has been the least understood step in spite of its importance in regulating the HO activity. In this study, we have performed reactions of a verdoheme-HO complex with alkyl hydroperoxides to assess three possible pathways previously proposed for the 123 S84 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

Direct electrochemistry of ascorbate oxidases on carbon brack modified electrodes has been investigated with cyclic voltammetry. The catalytic current was observed at the potential which was close to the formal potential of a type 1 Cu site at pH 7.0. On the other hand, in the absence of enzymes no catalytic current was observed. These facts indicate that the DET reaction occurs at the carbon black modified electrode and that the type 1 Cu site is a primary electron acceptor from the electrode. The dependences of the electrocatalytic currents on the solution pH, for values ranging from pH 4.0 to 8.0, have been investigated. The defference in the pH-optima for ASOM and ZAO was observed. ASOM and ZAO showed the pH-optima about 4.5 and 6.0, respectively. These pH-optima are good agreement with previously reported their own pH-optima in the reaction with ascorbate. In addition, the catalytic current obtained from ASOM modified electrode was higher than that from ZAO at each pH. This could reflect the stability of the enzymes on the electrode surface. However, further studies are required to define the precise causes of the difference in their activity. P153 Coordination Chemistry at the Fe(II) Site of Fe(II)/2- Ketoglutarate Dependent Hydroxylases P155 John L. McCracken1, Meng Li1, Piotr K. Grzyska2, Robert P. Zinc ion- and redox-regulated metamorphosis of Hausinger2, 1Department of Chemistry, Michigan State University, 2 oligomeric state for porphobilinogen synthase activation East Lansing, MI, USA; Department of Microbiology and Noriyuki Nagahara1, Nori Sawada1, Fumio Arisaka2, Kaoru Molecular Genetics, Michigan State University, East Lansing, MI, Mitsuoka3, Masayasu Minami1, 1Dept. of Envionmental Medicine, USA. Nippon Medical School, Tokyo, Japan; 2Graduate School of Contact e-mail: [email protected] Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan; 3Biological Information Research Center, One- and two-dimensional Electron Spin Echo Envelope National Institute of Advanced Industrial Science and Technology, Modulation (ESEEM) experiments have been used to study the Tokyo, Japan. coordination chemistry at the Fe(II) site of two non-heme Fe(II) Contact e-mail: [email protected] Hydroxylases, taurine/2-ketoglutarate (2KG) dioxygenase (TauD) and xanthine hydroxylase (XanA). For both systems, Fe(II)-NO Human porphobilinogen synthase (PBGS) [EC.4.2.1.24] catalyzes derivatives of the enzymes were studied. The 4-pulse HYSCORE the condensation of two molecules of 5-aminolevulinic acid to method was used to resolve contributions from bound histidine produce porphobilinogen. The oligomeric state of PBGS is nitrogens, coordinated H2O, and ambient H2O. For TauD, homooctamer, which consists of microheterogenous subunits in the HYSCORE spectra collected for samples in aqueous buffer and tertiary structure under air-saturated conditions. Two subunits form 60% D2O-buffer showed changes in H2O and histidyl coordination a minimal structural unit; in one subunit, Cys122, Cys124, and Cys132 as co-substrates, 2KG and taurine, were added to the enzyme. Prior near a catalytic sites coordinate a proximal zinc ion, which is to co-substrate addition, HYSCORE spectra show a substantial catalytically essential, and in the other subunit, Cys223 at the orifice distribution of exchangeable, 1H hyperfine couplings. When 2KG is of the active center coordinates a reservoir distal zinc ion. We added, the 1H HYSCORE is considerably altered with the dominant elucidated that, in enzymatic activation by 2-mercaptoethanol, the hyperfine coupling arising from an exchangeable, strong-coupled distal zinc ion was transferred, and bound to the proximal binding- proton of rhombic symmetry. Subsequent addition of substrate site (1). In this study, analytical ultracentrifugation, negative taurine to yield the ternary, Fe(II)-NO/2KG/taurine, complex at the staining electron microscopy, and native PAGE were performed to active site showed a new, 1H hyperfine interaction that was not determine the oligomeric state of PBGS under physiologic exchangeable in D2O. The HYSCORE cross-peaks from this 1H conditions. Furthermore enzyme activity staining and enzyme show a hyperfine tensor of axial symmetry characterized by a kinetic study were also carried out to investigate the structural dipole-dipole distance of 3.2A and an isotropic contribution of 1.1 change in enzymatic activation. It is concluded that PBGS exists in MHz. Comparison of these data with the X-ray crystal structure of octamer under physiological conditions, and that the metamorphosis TauD and the results of parallel studies of TauD variants, may of the four sets of dimer units, resulting from the decrease in the provide important information on how active site protein residues Stokes radius of the oligomer, occurs in enzymatic activation. work in concert with the Fe(II) center to catalyze the specific Metal- and redox-regulated metamorphosis of the oligomer is an hydroxylation of taurine. Parallel studies on XanA will be presented essential process for PBGS activation. and the results compared to our findings for TauD. (1) Sawada N, et al. (2005) J Biol Inorg Chem 10: 199-207

P154 P115a Direct electron transfer reactions of ascorbate oxidase on Iron-sulfur cluster biosynthesis in Arabidopsis thaliana carbon brack modified electrodes Masato Nakai, Institute for Protein Research, Osaka University, Kenichi Murata, Nobuhumi Nakamura, Hiroyuki Ohno, Suita, Japan. Department of biotechnology and Life Science, Tokyo University Contact e-mail: [email protected] of Agriculture and Technology, Tokyo, Japan. Contact e-mail: [email protected] Iron-sulfur proteins are found in virtually all life forms including archaebacteria, eubacteria, plants, and animals, and play various In the past few years, direct electron transfer (DET) reactions important physiological roles in electron transport, enzyme between electrodes and multicopper proteins have attracted much reactions, gene regulation, and DNA/RNA metabolisms. While a interest for construction of amperometric biodevices such as a number of factors involved in iron-sulfur cluster biosynthesis in biofuel cell and biosensors. Here, electrochemical studies of bacteria and in yeast have been identified and characterized ascrobate oxidases from zucchini (ZAO) and fungal Acremonium extensively, molecular mechanism of iron-sulfur cluster sp. HI-25 (ASOM) have been performed. biosynthesis and delivery in the plant cell still remains poorly 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S85

understood. In Arabidopsis thaliana, iron-sulfur cluster biosynthetic contains the nickel ion with the equatorial plane of monoamine- machineries have shown to be present both in the mitochondrion monoamide-dithiolate type coordination, and such a planar N2S2 and in the chloroplast. In the chloroplast, two essential factors have coordination geometry is necessary for the most suitable redox already been identified as important participants, namely CnfU and reaction of superoxide-dismutation. The crystal structural analysis Hcf101. Previously we identified and characterized the chloroplast- of NiSOD has showed that the central nickel ion is changed from localized iron-sulfur cluster biosynthetic scaffold named CnfU the square-planar Ni(II) to square pyramidal Ni(III) by coordination whose function is essential for biogenesis of ferredoxin and of axial imidazole of His1. photosystem I in Arabidopsis. We have now determined the crystal In this study, we focused on the axial coordination behavior in structure of CnfU and also gained the precise Fe-S cluster structure NiSOD, so three types of Ni(II) complexes with N2S2 square- assembled on this scaffold protein in order to elucidate detailed planar coordinations (Figure) as active site models of NiSOD were molecular mechanisms of CnfU-involved Fe-S cluster assembly and synthesized. Addition of to the acetone solution of Ni(II) delivery. Moreover, we have been characterizing several additional complexes, 1 and 2, showed absorption spectral changes due to their Arabidopsis proteins which are likely involved in iron-sulfur cluster axial coordination, but, in the case of Ni(II) complex 3, no spectral biosynthesis in the chloroplast or outside the chloroplast, the changes were observed under the same condition. EPR studies of an cytosol. We are now studying their detailed in vivo functions by oxidized Ni (III) complex of 3 revealed that an imidazole molecule analyzing Arabidopsis T-DNA insertion mutants and by using yeast bound to the axial position of the Ni(III) center, judging from the complementation assay. Biochemical characterization of respective appearance of hyperfine-splitting of its axially coordinating recombinant proteins purified from E.coli cells is also underway. nitrogen atom in the parallel region. Based on these spectroscopic analysis data of these model systems, we will discuss about relationships between the axial coordination behaviors and P156 electrochemical properties of the nickel center of NiSOD. Expression and characterization of iron containing alcohol dehydrogenase from hyperthermophilic archaeon, Pyrococcus horikoshii OT3 Yumi Kariya, Hirotoshi Matsumura, Nobuhumi Nakamura, Masafumi Yohda, Hiroyuki Ohno, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan. Contact e-mail: [email protected] P158 Alcohol dehydrogenases (ADHs; EC 1.1.1.1) belong to the oxidoreductase family, which catalyze the interconversion of The mechanism of arsenic binding to one and two- alcohols with the corresponding aldehyde or ketone, using the domain metallothioneins nicotinamide cofactors, NAD(H) or NADP(H). The NAD- or Thanh T. Ngu, Moira K. Rushton, Martin J. Stillman, The NADP-dependent ADH can be divided into three groups according University of Western Ontario, London, ON, Canada. to their structures. Group I is a zinc-dependent, long-chain ADH Contact e-mail: [email protected] family. Group II is a zinc-independent short-chain ADH. Group III is an iron-activated ADHs. The iron-activated ADHs have been The number of reported cases of chronic arsenic poisoning is on the little studied due to their structural instability. In this study, we have rise throughout the world, making the study of the long-term effects 3+ constructed the overexpression system of iron containing ADH of As exposure critical. As binds to biological thiols, including derived from hyperthermophilic archaeon, Pyrococcus horikoshii mammalian metallothionein (MT), which is an ubiquitous sulfur- OT3 (PhADH) with Escherichia coli. The catalytic activity and rich metalloprotein that coordinates a wide range of metals. The thermostability of PhADH have been investigated. two-domain mammalian protein binds divalent metals (M) into two PhADH gene was amplified by PCR using P. horikoshii OT3 metal-thiolate clusters with stoichiometries of M3Scys9 (ȕ) and 3+ genomic DNA. The gene subcloned into the pET23d was M4Scys11 (Į). We report that As binds with stoichiometries of transformed into E. coli BL21 (DE3). Cells were cultured at 37 °C As3Scys9 (ȕ) and As3Scys11 (Į) to the recombinant human in LB medium containing ampicillin and FeSO4. After metallothionein (rhMT) isoform 1a protein. Further, we report the centrifugation and sonication, the supernatant was heated to 80 °C complete kinetic analysis of the saturation reactions of the separate 3+ for 30 min. Objective protein was isolated via cation-exchange, Į and ȕ domains of rhMT with As . Speciation in the metallation hydrophobic, and gel filtration chromatographies. The purified reactions was determined using time- and temperature-resolved recombinant enzyme migrated as a single band with apparent electrospray ionization mass spectrometry (ESI-MS). The binding 3+ molecular mass about 43 kDa on SDS-PAGE, in good agreement reaction of As to the Į and ȕ MT domains is shown to be with that deduced from the gene sequence The substrate specificity noncooperative and involves three sequential, bimolecular of PhADH in the oxidation reaction was analyzed using a range of metallation steps. The time-dependent As-metalation to the two- primary alcohols. As a result, the highest catalytic activity of domain 16-cysteine seaweed Fucus vesiculosus metallothionien is PhADH using 1-butanol and NAD+ as a cofactor was obtained. The also described. The analyses allow for the first time, the complete catalytic activity of PhADH was maintained after pre-incubation at simulation of the experimental data for the step-wise metallation 90 °C. reaction of MT showing the relative concentrations of the metal- free, apo-MT and each of the As-MT intermediate species as a function of time and temperature. P157 1. Ngu, Thanh T. and M. J. Stillman (2006) Arsenic Binding to J. Am. Chem. Soc. Spectroscopic and electrochemical properties of Ni(II) Human Metallothionein. 128: 12473-12483 complexes with axial coordination as an active site model 2. Merrifield, M.E., Ngu, T., Stillman, M.J. (2004) Arsenic binding to Fucus vesiculosus metallothionein. Biochem. Biophys. Res. of NiSOD. Commun. 324: 127-132 Daisuke Nakane, Tatsuya Fujii, Yasuhiro Funahashi, Tomohiro Ozawa, Hideki Masuda, Graduate school of materials and engineering, Nagoya, Japan. Contact e-mail: [email protected]

Ni-containing superoxide dismutase (NiSOD) is an enzyme catalyzing disproportionation of superoxide anion through the cycles of Ni(II) and Ni(III) states. The active site of NiSOD 123 S86 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

P159 HemAT-Rr have three conformers but the hydrogen-bonding Arylhaloallylamine Complexes of Copper-Containing pattern in the distal heme pocket is different from HemAT-Bs. Amine Oxidases Based on these results, we will discuss the essential factors for the Yen H. Nguyen1, David B. Langley1, Ping Ying2, David M. selective O2 sensing by HemAT Dooley3, Hans C. Freeman1, Ian A. McDonald2, J. M. Guss1, 1University of Sydney, Sydney, Australia; 2Pharmaxis Ltd., Frenchs Forest, Australia; 3Montana State University, Bozeman, MT, USA. P161 Contact e-mail: [email protected] Spectroscopic and Electrochemical Studies of Pseudoazurin Met16X Variants Semicarbazide-sensitive amine oxidase (SSAO, AOC-3) is a Yuji Obara1, Doreen E. Brown2, David M. Dooley2, Takamitsu member of the family of copper-containing amine oxidases and Kohzuma1, 1Ibaraki Univ., Ibaraki, Japan; 2Montana State Univ., encompasses a variety of roles that include detoxification in Bozeman, MT, USA. mammals, wound healing in plants, and provision of essential Contact e-mail: [email protected] nutrients in bacteria. The human SSAO, also called vascular adhesion protein-1 (VAP-1), is of particular interest, owing to its Noncovalent weak interactions play important roles in many adhesion and enzymatic properties that have been proven to be varieties of biological systems. Very recently, we reported the implicated in inflammation-related diseases, such as rheumatoid spectroscopic and electrochemical studies of M16F, M16Y, and arthritis, multiple sclerosis, Crohn’s disease, diabetes, and M16W mutants of blue copper protein, pseudoazurin (PAz) to atherosclerosis. investigate the effects of the ʌ-ʌ interaction on the active site We are undertaking structural studies of various arylhaloallylamine structure [1]. The Met16 substituted mutants of PAz, in which derivatives with Arthrobacter globiformis amine oxidase (AGAO), several alkyl group has been introduced in the vicinity of the His81 which possesses structural and enzymatic similarities to VAP-1. imidazole ligand, have been constructed and characterized in order These inhibitors have been previously reported to be mechanism- to elucidate the effects of the indirect weak interaction on the based inhibitors of monoamine oxidase (MAO) and SSAO. These structure and function of the active site. Electronic absorption compounds consist of a primary amine moiety which poses as the spectra of M16I, M16V, M16A, M16L mutants and WT showed the substrate and allows the inhibitors to form a Schiff-base absorbance ratio, A~460/A~600 were 0.70, 0.61, 0.60, 0.43, and 0.46 intermediate with the topaquinone cofactor (TPQ, a modified respectively. The ratio values except M16L variant are significantly tyrosine residue) during the catalytic cycle. This hypothesis is higher than that of WT PAz. The higher value suggests the supported by preliminary data indicating the inhibitor-TPQ adduct increasing of rhombic structure population. is trapped in the active site. A part of this work was supported by the Research Promotion Bureau, Ministry of Education, Culture, Sports, Science, and Technology, Japan, to T.K., under contract no. 17-214. Reference [1] Rehab F. Abdelhamid. et. al., J. Biol. Inorg. Chem, 12 (2), 165 (2007).

P162 Rate limiting event for Paracoccus pantotrophus CCP P160 catalysis as revealed by direct electrochemistry Oxygen sensing mechanism of signal transducer protein Patricia M. Paes de Sousa1, Sofia R. Pauleta1, David Rodrigues2, HemAT M. Lurdes Simões Gonçalves2, Graham W. Pettigrew3, Isabel Muneto Nishimura1,2, Hideaki Yoshimura1,2, Kazumichi Ozawa2, Moura1, Margarida M. Correia dos Santos2, José J. G. Moura1, Shiro Yoshioka2, Minoru Kubo2, Teizo Kitagawa2, Shigetoshi 1REQUIMTE-CQFB, FCT-UNL, Monte da Caparica, Portugal; Aono2, 1The Graduate University for Advanced Studies, Okazaki, 2CQE-IST, Lisboa, Portugal; 3Royal (Dick) School of Veterinary Japan; 2Okazaki Institute for Integrative Bioscience, Okazaki, Studies, University of Edinburgh, Edinburgh, United Kingdom. Japan. Contact e-mail: [email protected] Contact e-mail: [email protected] Reactive oxygen species like hydrogen peroxide are toxic HemAT is a heme-containing signal transducer protein that compounds leading to cell damage or death. Peroxidases reduce functions as an O2 sensor in aerotaxis control system in some peroxide to H2O using a variety of oxidisable substrates, small bacteria and archaea. HemAT should discriminate its physiological mono haem c type cytochromes and/or copper proteins, like effecter O2 from other gas molecules, for which the specific pseudoazurin [1,2]. interaction between the heme-bound ligands and the surrounding As to the catalytic mechanism, with the exception of Nitrosomonas amino acid residues play an important role. Resonance Raman europaea, all bacterial cytochrome c peroxidases (CCP) are isolated spectroscopy and mutagenesis studies reveal that HemAT from in a resting state. The high potential haem needs to be reduced by Bacillus subtilis (HemAT-Bs) has three conformers in O2-bound one electron to allow the conversion from resting to active protein, form with different hydrogen bonding pattern on the O2, where triggering the loss of one of the coordinants from the low potential Thr95 is involved in the hydrogen bonding interaction. haem allowing the hydrogen peroxide to bind. Recently, it is reported that HemAT homologues exist in some We report on the direct electrochemistry of Paracoccus bacteria. In some HemAT homologues, Thr residue is not conserved pantotrophus CCP in non turnover and catalytic conditions at a at the corresponding position to Thr95 in HemAT-Bs in the distal pyrolytic graphite membrane electrode. The results show that the heme pocket. This suggests that the hydrogen bonding interaction enzymatic activity is mediated and limited by the structural and spin between Thr residue and the heme bound O2 is not a mandatory state changes, following the electron transferring haem reduction. requirement for the selective O2 sensing mechanism. Essential The estimated turnover number is in clear agreement with the rate factors for O2 sensing by HemAT would be clarified by comparing constant determined for the rate limiting step. the detail properties including the hydrogen bonding pattern on the [1] Pauleta SR, Guerlesquin F, Goodhew CF, Devreese B, Van heme-bound ligand among HemAT homologues. Beeumen J, Pereira AS, Moura I and Pettigrew GW, Biochem, In this study, we characterized the hydrogen bonding pattern on the 2004, 43, 11214-11225. heme-bound O2 in HemAT from Halobacterium salinarum [2] Paes de Sousa PM, Pauleta SR, Simões Gonçalves ML, (HemAT-Hs) and Rhodospirillum rubrum (HemAT-Rr) by using Pettigrew GW, Moura I, Correia dos Santos MM*, Moura JJG, J resonance Raman spectroscopy. O2-bound HemAT-Hs and Biol Inorg Chem, 2007, accepted. 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S87

[3] Pettigrew GW, Echalier A, Pauleta SR, J Inorg Biochem, 2006, As another of our current interests lies in the redox state controlled 100, 551-567. mobilization of metal ions in plant MTs, we will further compare II Acknowledgments: This work is within the research project Zn -release rates from Ec-1 and other plant MT sub-types under POCI/QUI/55743/2004. P. Paes de Sousa, S. R. Pauleta and D. varying redox conditions with each other. Rodrigues thank FCT for financial support. [1] W. Maret, J. Nutr. 2000, 130, 1455S-1458S [2] B. Lane, R. Kajioka, T. Kennedy, Biochem. Cell. Biol. 1992, 65, 1001-1005 P163 [3] E. A. Peroza, E. Freisinger, J. Biol. Inorg. Chem. 2007, DOI Microwave-Assisted Solid-Phase Synthesis, Cellular 10.1007/s00775-006-0195-5 Uptake Studies, and Cytotoxicity of Cymantrene-Peptide Support from the Swiss National Foundation (200020-113728/1 to Bioconjugates E.F.) is gratefully acknowledged. Harmel Wilfried P. Peindy Ndongo, Ulrich Schatzschneider, Lehrstuhl für Anorganische Chemie I – Bioanorganische Chemie, P165 Ruhr-Universität Bochum, Universitätss, Bochum, Germany. Contact e-mail: [email protected] Does the deprotonation of CuB ligands play a role in reaction mechanism of Cytochrome c oxidase? Incorporation of metallocenes into biological molecules has M. Punnagai, G. Matthias Ullmann, University of Bayreuth, attracted the attention of several researchers. Thus, steroids, Bayreuth, Germany. penicillin antibiotics, and saccharides have been functionalized with Contact e-mail: [email protected] metallocenes. The rationale to prepare such bioconjugates lies in the increased lipophilicity brought by the metallocene structure and the Recent studies on the cytochrome c oxidase (CcO) CuB center use of the covalently bound metal-label for sensing applications. suggested that the deprotonation of the CuB bound imidazole ring Despite many advances in peptide chemistry and the synthesis of of histidine (His291 in mammalian CcO or His284 in Rhodobacter peptide-metallocene bioconjugates, cymantrene derivatives have sphaeroides CcO) as a key element in the proton pumping received relatively little attention so far. mechanism. The central feature of the proposed mechanism is that In this work, the neuropeptide pseudoneurotensin H-Lys-Lys-Pro- pKa values of the imidazole vary significantly depending on the Tyr-Ile-Leu-OH, a 99mTc-labelled analogue of which shows redox state of the metals in the binuclear center. We report the pKa enhanced tumor uptake, was chosen to be part of metal-peptide values of the His291, His290 and water molecules both in the bioconjugates. We report on the microwave-assisted solid phase oxidized and reduced state of CuB center, where the the pKa values conjugation between cymantrene carboxylic acid and the of the His291 was considered to play a significant role in the biologically active neuropeptide part (Scheme). Microwave reaction mechanism of cytochrome c oxidase. We use hydrid irradiation was applied at different stages of the synthesis, including density functional theory in combination with continuum amide bond formation, removal of Fmoc protective groups, and electrostatics to calculate the pKa values, successively with aqueous acidolytic release of the metal-bioconjugate from the resin. The use solution and with in the protein, of the Cu-bound imidazoles in of microwave irradiation in all steps of the solid-phase peptide oxidized and reduced states respectively. In the present study we synthesis and for coupling of cymantrene allows to significantly considered all the possible protonation equilibrium reactions in the reduce reaction times while leading to high purity of the crude CuB center to understand the deprotonation reactions of His290, product. The resulting metal-peptide bioconjugate (1) was purified His291 and water. The pKa shifts are calculated in protein by reverse-phase HPLC and characterized with ESI mass environment. spectrometry. Additionally, the biological activity of this new 1. D. M. Popovic, J. Quenneville, A. A. Stuchebrukhov, J. Phys. cymantrene-peptide will be reported. Chem. B 2005, 109, 3616- 3626. This work was supported by the Deutsche Forschungsgemeinschaft 2. E. Fadda, N. Chakrabarthi, R. Pomès, J. Phys. Chem. B 2005, (DFG) within FOR 630 (,,Biological Function of Organometallic 109, 22629-22640. Compounds”) through grant SCHA 962/3-1. 3. G. M. Ullmann, L. Noodleman, D. A. Case, J. Biol. Inorg. Chem. 2002, 7, 632-639. 4. G. M. Ullmann, J. Phys. Chem. B 2003, 107, 1263-1271.

P166 P164 The complex III from Rhodothermus marinus Wheat Ec-1 MT: A detailed view and a comparison with Manuela M. Pereira1, Patrícia N. Refojo1, Gudmundur O. other plant metallothioneins Hreggvidsson2, Sigridur Hjorleifsdottir2, Miguel Teixeira1, Estevão Peroza, Eva Freisinger, Institute of inorganic chemistry, 1Instituto de Tecnologia Química e Biológica, Oeiras, Portugal; Zurich, Switzerland. 2Prokaria Ltd, Sudurgata, Iceland. Contact e-mail: [email protected] Contact e-mail: [email protected] Metallothioneins (MTs) are small proteins found in a wide range of living organisms with a characteristic high cysteine content and Rhodothermus marinus is a thermohalophic bacterium, whose 10 affinity for d metals ions. There is evidence supporting their respiratory chain has been extensively studied. The biochemical, II participation in the homeostasis of essential metal ions, e.g. Zn and spectroscopic and genetic search for a bc complex was always I II II 1 Cu , sequestration of toxic elements (Cd or Hg ) and in the fruitless; however a functional equivalent complex was purified protection against oxidative damage. Additionally, the mobility of from the membranes and characterized1. Now, with the sequencing metabolic zinc has been linked to the cellular redox state and thus of R. marinus genome it was possible to assign the N-terminal also to the redox state of MTs [1]. sequences from several proteins of this complex to its coding genes. The early cysteine-labelled protein (Ec-1) from wheat is a member It was observed that the bc complex has the same genomic of the plant MT pec sub-family and was the first MT identified in organisation of the so called MFIcc complexes2, which have been higher plants [2]. In our past work we showed that wheat Ec-1 binds proposed to be participating in the respiratory and II II up to 6 Zn or Cd ions. These metal ions are most likely arranged in the photosynthetic electron transfer chains. Furthermore it was in two separate metal clusters, one binding four metal ions (Į-Ec-1) observed the presence of this complex in several genomes in which and another binding two ( -E -1) [3]. In our contribution we will Ȗ c the genes coding for the bc1 complex are absent and in which a present a more detailed view of the metal cluster characteristics. quinol:cytochrome c oxidoreductase has to be present. R. marinus bc complex is coded by a six gene cluster. Three of these genes 123 S88 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

codify for peripheral proteins; two cytochromes c, a pentahaemic this complex is reacted with oxygen for 2 s, a reaction intermediate and a monohaemic, and a large protein containing iron sulphur is formed in high yield (80%). Mössbauer spectroscopy centres. The other three genes code for transmembrane proteins: demonstrates that the novel species contains a high-spin Fe(IV) two are predicted to have ten transmembrane helices with putative center. The enhanced preedge feature in the Fe-X-ray absorption quinone binding sites motifs and are homologous to membrane spectra and the short Fe-oxygen and Fe-Br interactions of 1.62 Å subunits present in several members of the DMSO reductase family; and 2.43 Å, respectively, support the formulation of this the third gene codifies a two transmembrane helices protein. intermediate as a Br-Fe(IV)-oxo complex. 1 Pereira, M. M., Carita, J. N and Teixeira, M (1999) Biochemistry, (1) Ueki, et al., Chem. Biol, 2006, 13, 1183-1191 38, 1268-1275 (2) For a recent review, see Vaillancourt, et al., Chem. Rev. 2006, 2 Yanyushin, M. F., del Rosario, M. C. Brune, D. C and 106, 3364-3378. Blankenship, R. E. (2005) Biochemistry 44, 10037-10045 (3) Galonic, et al., Nature Chemical Biology 2006, 3, 113-116.

P167 P169 Identification and analysis of cadmium protein targets in Metal ion binding and structural dynamics of human Escherichia coli : a 2D electrophoresis approach hemopexin maintaining protein-metal interactions performed in vivo Federico I. Rosell, Marcia R. Mauk, A. Grant Mauk, Dept. of Stéphanie Renaud1, Nicolas Bremond1, Céline Henry2, Catherine Biochemistry and Molecular Biology and Centre for Blood Berthomieu1, 1LIPM, IBEB, CEA cadarache, Saint Paul Lez Research, University of British Columbia, Vancouver, BC, Canada. Durance, France; 2PAPSS, BIOBAC, INRA, Jouy en Josas, France. Contact e-mail: [email protected] Contact e-mail: [email protected] The heme-scavenging plasma protein hemopexin (Hx) binds a Despite numerous studies, the molecular mechanism of cadmium variety of divalent metal ions1 and may, therefore, participate in (Cd) toxicity in cells is not totally understood. Our aim is to detect metal ion homeostasis in vivo. Recently, we used homology protein targets of Cd at low Cd doses (10 μM) to unravel some modeling to propose a structure for human Hx based on the aspects of cadmium toxicity. Although Cd-protein interactions have structure reported for rabbit Hx, and we identified several putative been evidenced in vitro, only few data are available concerning metal ion binding sites.2 Here, we use thermally-induced near-UV these interactions in vivo, at low Cd doses. difference spectroscopy to study human Hx lacking bound heme To identify Cd protein targets in vivo, cells were grown in the (the form normally in circulation) in the presence and absence of presence of radiolabeled 109Cd. Two successive native bound metal ions. The results are interpreted in light of the metal electrophoreses were performed to separate solubles proteins of the ion binding sites proposed in our model.2 We also consider the cell extract. This method preserves metal-protein interactions effects of increasing temperature and metal ion binding on the far- established in vivo. Autoradiography revealed fourteen soluble UV CD spectrum of Hx and find that Zn2+ and Cu2+ influence the protein targets for cells cultured in the presence of 10 μM Cd. tertiary structure of the protein the most by reducing the Tm by 4 Proteins were then identified using mass spectrometry. Mutants and 5 °C, respectively. Because these metal ions also destabilize the deleted for some these proteins were constructed which confirmed structure of the Hx-heme complex,3 we studied their effect on the the identified protein targets. Overexpression of some of the target spectroscopic properties and CO recombination kinetics of the Hx- proteins confirmed their role in Cd-binding in the cellular context. Fe(II)-heme complex. These results are consistent with binding of This work validates our new global approach for protein metal Cu2+ in the linker region between the two domains of the protein as targets identification. recently proposed. Supported by a CIHR/CBS partnership grant and Analysis of the consequence of Cd binding on the proteins structure a Canada Research Chair (to A.G.M.). and function has been initiated. In particular, Cd substitution of 1. Mauk, M. R.; Rosell, F. I.; Lelj-Garolla, B.; Moore, G. R.; Mauk, endogenous metals (Fe/Zn) is being analyzed using FTIR difference A. G. Biochemistry 2005, 44, 1864-1871. spectroscopy (see poster by L. Guilloreau et al.). 2. Mauk, M. R.; Rosell, F. I.; Mauk, A.G. Nat. Prod. Rep. in press. 3. Rosell, F. I.; Mauk, M. R.; Mauk, A.G. Biochemistry 2005, 44, 1872-1979. P168 Characterization of an iron(IV) intermediate from the P170 Fe(II)- and alpha-ketoglutarate-dependent halogenase CytC3 NMR analysis for association and dissociation Pamela Riggs-Gelasco1, Danica P. Galonic2, Eric W. Barr3, Megan mechanisms between Cytochrome c and Cytochrome c L. Matthews3, Gretchen M. Koch3, Joseph R. Yonce1, Joseph M. Oxidase Bollinger3, Christopher T. Walsh2, Carsten Krebs3, 1College of Koichi Sakamoto1, Masakatsu Kamiya2, Kyoko Shinzawa-Itoh3, Charleston, Charleston, SC, USA; 2Harvard Medical School, Tomoyasu Aizawa4, Makoto Demura2, Keiichi Kawano4, Shinya Cambridge, MA, USA; 3The Pennsylvania State University, Yoshikawa3, Koichiro Ishimori1, 1Division of Chemistry, Graduate University Park, PA, USA. School of Science, Hokkaido University, Sapporo, Japan; 2Division Contact e-mail: [email protected] of Life Science, Graduate School of Life Science, Hokkaido University, Sapporo, Japan; 3Department of Life Science, Graduate The enzyme CytC3 from Streptomyces halogenates the Ȗ-methyl School of Life Science, University of Hyogo, Ako, Japan; group of L-aminobutyric acid to produce 4-chloro and 4, 4 - 4Department of Biological Sciences, Graduate School of Science, dichloroaminobutyric acids, of which the latter shows antibacterial Hokkaido University, Sapporo, Japan. activity.(1) To achieve halogenation of the unactivated methyl Contact e-mail: [email protected] carbon, CytC3 and other non-heme iron halogenases utilize Į- c c ketoglutarate in an O2 dependent reaction to generate a high-valent Cytochrome (Cyt ) is a key electron mediator between Fe(IV)-oxo species. The intermediate abstracts a hydrogen atom Cytochrome bc1 complex and Cytochrome c Oxidase (CcO) in the from the substrate, allowing the incorporation of a metal-bound respiratory chain. To properly transfer electrons from Cyt c to CcO halide into the substrate radical.(2,3) We describe here the structural for the oxygen reduction in CcO, the specific and effective electron characterization of the CytC3 Fe(IV)-oxo intermediate that was transfer mechanism is required. The detailed mechanism is, generated in the presence of bromide rather than chloride using x- however, still unclear due to lack of the structural characterization ray absorption and Mössbauer spectroscopies. Iron x-ray absorption of the electron transfer complex between Cyt c and CcO. In this spectroscopy demonstrates an Fe-Br interaction in the reactant study, 1H-15N HSQC NMR spectra of ferrous and ferric 15N-labeled CytC3:Fe(II):ĮKG:Br-:L-Aba-S-CytC2 complex of 2.51 Å. When Cyt c were measured in the absence and presence of unlabeled CcO 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S89

to examine the CcO binding site on Cyt c. Based on the chemical zinc ion analysis, native-PAGE and analytical ultracentrifugation shift changes and line broadening of the NMR signals, we were performed. successfully determined that the CcO binding site is located near The kcat and kcat/Km values for C223A were similar to those of wild heme vinyl group and showed the possibility that, in addition to type PBGS. When His131 was substituted with Ala or Arg, the electrostatic interactions, hydrophobic interactions are also involved kcat/Km values were decreased to 1/10 or 1/17, respectively. The in the specific binding with CcO. Upon oxidation of ferrous Cyt c to profiles of pH-dependency of kcat showed that the optimum pH and ferric Cyt c, the signal perturbation by binding CcO is reduced for pKa values were shifted toward alkaline pH. Zinc contents of wild some NMR signals from polar residues such as Glu4 and Lys8, type PBGS, H131A and C223A were 1, 1 and 0.5 Zn/subunit, suggesting that the electrostatic interactions with CcO would be respectively. The Zn concentration dependence of enzymatic weakened to facilitate the replacement of ferric Cyt c with ferrous activities revealed that Cys223 contributed to maintenance of the Cyt c. Thus, our NMR analysis for the Cyt c-CcO complex clearly catalytic efficiency at low concentration of zinc ion in the reaction identified the CcO binding site on Cyt c and we can propose a mixture. Native-PAGE showed that a structural change, which was mechanism for the redox-dependent affinity change of Cyt c to required for enzymatic activation by 2-mercaptoethanol and zinc CcO, leading to the specific and effective electron transfer between ions, was not observed in H131A. These findings suggest that His131 Cyt c and CcO. contributes to the structural change in enzymatic activation without coordination of zinc ions. P171 Iminodiacetic Carboxylate/Hydroxamate-Sulfonamide P173 Derivatives as Potential Dual Target Antitumor Drugs The catalytic mechanism of purple acid phosphatases Maria Amelia Santos, Centro de Química Estrutural, Lisboa, Nata a Mitiü1, Robynn S. Cox2, Maurizio Lanznaster3, Lawrence Portugal. R. Gahan1, Ademir Neves3, Alvan C. Hengge2, Gerhard Schenk1, Contact e-mail: [email protected] 1The University of Queensland, St Lucia, Australia; 2Utah State University, Logan, UT, USA; 3Universidade Federal de Santa Inhibitors of Zinc-containing enzymes, including Matrix Catarina, Florianópolis, Brazil. Metalloproteinases (MMPs) and some tumor-associated Carbonic Contact e-mail: [email protected] Anhydrases (CAs) isoenzymes are attractive therapeutic targets in the treatment of numerous diseases. Therefore their inhibition is Purple acid phosphatases (PAPs) belong to the family of binuclear supposed to slow down or prevent disease progression. The metallohydrolases that catalyze the hydrolysis of numerous inhibition of each of these enzymes needs special structural phosphoester substrates at acidic pH.1 PAPs from different sources requirements, not only in corresponding zinc-binding groups but (mammalians, plants, fungal) have been isolated and characterized, also in the different scaffolds to assure strong interaction within the and generally have an Fe(III) and a M(II) ion in the active site (M = cofactor-binding region of the enzymes. Fe, Mn, Zn). Despite low overall sequence homology between A series of new compounds, based on iminodiacetic (IDA) scaffolds kingdoms structural conservation in their active sites is observed. with different sets of alkylaryl and sulfonylaryl groups as well as The seven metal-coordinating residues are invariant. The precise two types of zinc binding groups, namely hydroxamate and primary details of the catalytic mechanism employed by PAPs remains sulfonamides have been designed, developed and tested in vitro, subject to debate.1 Here, we investigate the mode of substrate with the aim of improving potency and selectivity for the inhibition binding in pig PAP (uteroferrin) using stopped-flow spectroscopy, of some tumor-associated MMPs and some CA isoenzymes. These and we demonstrate, for the first time, that PAPs are also effective non-peptidic compounds were easily prepared, mostly from IDA, diesterases. Specifically, interactions between the leaving group and and they were assayed as inhibitors of seven MMPs (MMP1, residues in the second coordination sphere are essential for the rapid MMP2, MMP7-9, MMP14, and MMP16 and three CAs (hCA I, II and efficient binding of the substrate to both metal ions in the active and IX). Some of the compounds show excellent specificity for the site. Furthermore, using a diester substrate we demonstrate that PAP CA IX as well as MMP2, MMP9 and MMP13 with IC50 values in sequentially hydrolyses both ester bonds without releasing the nanomolar order. initially formed monoester. The most likely mechanism is initial attack by a terminal Fe(III)-bound hydroxide and then a metal- bridging hydroxide as nucleophile. A similar mechanism has also been proposed for the [L(OH2)Fe(ȝ-OH)Zn](II) PAP mimetic (where L = 2-bis[{(2-pyridyl-methyl)-aminomethyl}-6-{(2- hydroxy-benzyl)-(2-pyridyl-methyl)}-aminomethyl]-4-methyl- phenol). Mechanistic implications are discussed. 1 P172 Mitic, N.; Smith, S. J.; Neves, A.; Guddat, L. W.; Gahan, L. R.; Schenk, G. Chem. Rev. 2006, 106, 3338 Histidine131 is essential for metal- and redox-regulated activation of human porphobilinogen synthase Nori Sawada1, Noriyuki Nagahara1, Fumio Arisaka2, Yoko Endo3, Yoshiaki Nakajima3, Masayasu Minami1, Tomoyuki Kawada1, 1Dept. of Environmental Medicine, Nippon Medical School, Tokyo, Japan; 2Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan; 3Clinical Research Center on Occupational Poisoning, Tokyo, Japan. Contact e-mail: [email protected]

Human porphobilinogen synthase (PBGS) [EC.4.2.1.24] has two zinc binding sites; one site, referred to as a proximal zinc-binding site, consists of three cysteines near two catalytic sites. The other site, referred to as a distal zinc-binding site, consists of His131 and Cys223, which are located at the orifice of the catalytic cavity. To elucidate the role of His131 and Cys223 in the catalysis, expression vectors for wild type PBGS, H131A (His131 ĺ Ala), H131R, and C223A were constructed. Each protein was then overexpressed in E. coli and purified to homogeneity. Kinetic study, 123 S90 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

P174 References. Isolation, characterization, and monooxygenase activity [1] T. Kohzuma and S. Suzuki, Mol.Cryst.Liq.Cryst., 286, 95 of the minimal functional unit of octopus hemocyanin (1996). Chizu Shimokawa1, Kenji Suzuki1, Shigetoshi Aono2, Shinobu [2] I. Harada and H. Takeuchi in “Advances in Spectroscopy”, R. J. Itoh1, 1Department of Chemistry, Graduate School of Science, H. Clark, R. E. Hester eds., John Wiley& Sons, 13, 113 (1986). Osaka City University, Osaka, Japan; 2Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Japan. P176 Contact e-mail: [email protected] Drastic alteration of the substrate specificity based on the substrate misrecognition of cytochrome P450BSȕ Hemocyanin and tyrosinase are the members of type-3 copper Osami Shoji1, Takashi Fujishiro1, Takuya Hirose1, Hiroshi proteins, which involve a common side-on peroxo species at their Nakajima1, Misa Kim2, Shingo Nagano2, Yoshitsugu Shiro2, dinuclear copper reaction center (Chart 1). Despite having the same Yoshihito Watanabe1, 1Graduate School of Science, Nagoya side-on peroxo dicopper(II) complex, these copper proteins exhibit University, Nagoya, Japan; 2RIKEN SPring-8 Center, Harima different chemical reactivity toward external substrates. Namely, Institute, Sayo, Japan. tyrosinase catalyzes the o-hydroxylation of phenols to catechols Contact e-mail: [email protected] (phenolase activity) as well as the oxidation of catechols to o- quinones (catecholase activity). In contrast, hemocyanin has Cytochrome P450BSȕ (P450BSȕ) isolated from Bacillus subtilis essentially no redox reactivity toward external substrates, but utilizes hydrogen peroxide to catalyze exclusively the hydroxylation exhibits only reversible dioxygen binding ability. We have recently of long alkyl-chain fatty acid such as myristic acid. The X-ray found that octopus hemocyanin exhibits efficient monooxygenase crystal structure shows that the interaction of the terminal carboxyl activity (phenolase activity) when it is treated with 8 M urea (J. Am. group of the fatty acid with Arg242 located nearby the heme is Chem. Soc. 2006, 128, 6788-6789). In order to get more detailed indispensable for the generation of the active species. Owing to this insight into the monooxygenase activity of octopus hemocyanin, we unique catalytic mechanism, P450BSȕ never oxidizes substrates herein examined the reactivity of the minimal functional unit, except for the natural substrate. However, the substrate specificity containing a single dicopper active site, existed at the C-terminal of P450BSȕ was altered drastically by the misrecognition of the domain of the large subunit (~350 kDa). The expected minimal substrate induced by a decoy molecule having the structural functional unit with 47 kDa molecular weight was obtained by the similarity to that of the natural substrate such as heptanoic acid. In treatment of native octopus hemocyanin with V8-protease and the presence of the decoy molecule, P450BSȕ started to catalyze following column chromatographic treatments using Sephacryl S- oxidation reactions of non natural substrates including 100 HR. We have also tried to develop a gene expression system of hydroxylation of ethylbenzene (Scheme 1).1 The X-ray crystal the minimal functional unit of octopus hemocyanin to study the structure of P450BSȕ including heptanoic acid showed that heptanoic molecular mechanism of the monooxygenation activity. acid are located in the heme pocket with the interaction between its carboxyl group and Arg242 similarly to the natural substrate and it keeps the substrate access channel open for the access of non- natural substrates.

P175 UV Resonance Raman Spectroscopic Studies of Cytochrome c’ from Alcaligenes xylosoxidans NCIMB 11015 Naoko Shiono, Institute of Applied Beam Science, Mito,Ibaraki, Japan. Contact e-mail: [email protected]

Cytochromes c’ are a class of c-type cytochrome, which consists of four Į-helix bundle structure. Cyt c’ has been considered to be a NO binding protein in the denitrifying systems. Cyt c’ showed a well defined quasi-reversible faradaic response at a 6-mercaptopurin/Au Ref. (1). O. Shoji, T. Fujishiro, H. Nakajima, M. Kim, S. Nagano, electrode, and the redox potential of cyt c’ was evaluated to be 184 Y. Shiro, Y. Watanabe, Angew. Chem., Int. Ed. in press. mV [1]. The pH and ionic strength dependency of the structure and properties of cyt c’ has been reported. UV resonance Raman (UVRR) spectroscopic measurements of cyt c’ (ex. at 244 nm) were P177 performed to elucidate the pH and ionic strength effect on the Comparative study of energy metabolism in Bilophila structural transition of cyt c’. Most of prominent Raman bands are wadsworthia and an environmental sulfate-reducing readily assigned based on the previous paper [2]. The Raman bands bacterium at 1621 (Y8a), 1208 (Y7a), and 1175 (Y9a) cm-1 are contributed Sofia Silva, Filipa Valente, Inês Pereira, Instituto de Tecnologia from tyrosine residues, and the Raman bands at 1551(W3) and Química e Biológica, Oeiras, Portugal. 1343/1358(W7; Fermi doublet) cm-1 are contributed from Contact e-mail: [email protected] tryptophan residues [3]. UVRR spectra of cyt c’ at different pH values indicated that the environment of tryptophan residues was Bilophila wadsworthia is a Gram-negative bacterium and a member affected by pH. The effect of ionic strength on the UVRR of cyt c’ of the human gut microbial ecosystem. This bacterium is isolated will be reported and discussed. from a variety of pathogenic infections, being the third most This work was supported by the Project of Development of Basic common anaerobe present in patients with perforated and Technologies for Advanced Production Methods Using gangrenous appendicitis. Although related to Desulfovibrio Microorganism Functions by the New Energy Development organisms, which are sulfate reducers, it has a particular Organization (NEDO). metabolism adapted to the human body since it uses taurine, an 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S91

organosulfonate that is one of the major solutes in mammals. and hydrogen peroxide, respectively. The well-characterized Desulfovibrio vulgaris Hildenborough is an environmental intermediate, diferric peroxo, of maxi-ferritins appeared to form bacterium that is a model organism for sulfate reducers. Its genome, anerobically in mini-ferritin Dps 2 (Liu et al, JBC, 281: 27827, which was recently sequenced, codes for several hydrogenases and 2006), although the putative iron ligands are very different. These formate dehydrogenases, which are enzymes that play an important data provide new insight into the structure/reactivity of di-iron role in Desulfovibrio energy metabolism. Since Bilophila oxidase sites and formation of ferric mineral precursors. wadsworthia is a pathogen whose physiology is still poorly understood, we performed a comparative activity study of these enzymes in both organisms to achieve a better understanding of the P180 energy metabolism of B. wadsworthia. Gas-sensing hemeproteins in Mycobacterium tuberculosis: a role for oxygen in pathogenesis Eduardo H. S. Sousa, Jason R. Tuckerman, Gonzalo Gonzalez, P178 Marie-Alda Gilles-Gonzalez, University of Texas Southwestern EPR and ENDOR characterization of pMMO from Medical Center, Dallas, TX, USA. Methylococcus capsulatus (Bath) and Methylosinus Contact e-mail: [email protected] trichosporium OB3b. Stephen M. Smith1, Amanda S. Hakemian1, Joshua Telser1,2, Brian During this past decade, heme-based sensor proteins have been M. Hoffman1, Amy C. Rosenzweig1, 1Northwestern University, discovered in all kingdoms of life at a very fast pace. These sensors 2 Evanston, IL, USA; Roosevelt University, Chicago, IL, USA. usually bind O2, CO and NO while engaging in signal transduction Contact e-mail: [email protected] with one of these gases. FixL was the first heme-based sensor discovered and represents a paradigm. Symbiotic rhizobia, upon Particulate methane monooxygenase (pMMO) from Methylococcus invasion of the plant root, face much lower oxygen tensions. FixL- capsulatus (Bath) and Methylosinus trichosporium OB3b is a FixJ are responsible for sensing oxygen levels and promoting gene copper-containing enzyme that converts methane to methanol. As- expression for bacterial adaptation. Similar behavior could be isolated pMMO from both species contains a mixture of Cu(I) and assumed for Mycobacterium tuberculosis (Mtb). Mtb entrance into Cu(II). Crystallographic characterization of both pMMOs reveals latency is thought to be linked to hypoxia in the granuloma, and the presence of several metal centers, but details of the also, recently, to NO. Genetic and biochemical experiments have coordination, including the possible presence of exogenous ligands, supported that the main regulator of hypoxia-induced genes of Mtb remain unclear. In addition, the active has not been identified. In is the response regulator DevR. Two histidine kinases called DosT this study, the copper centers in highly active crude pMMO and DevS are responsible for DevR activation. In 2005, DevS was membranes and purified pMMO samples from M. capsulatus (Bath) shown to contain heme, but there was no characterization or and M. trichosporium OB3b were investigated by electron functional investigation of the holo protein. We have expressed and paramagnetic resonance (EPR) and 1H, 15N, and 19F electron nuclear characterized full-length holo DosT and DevS proteins. DosT is double resonance (ENDOR) spectroscopies. The EPR spectra show also a heme containing protein. Both proteins present very similar a typical type 2 Cu(II) signal for both pMMOs that accounts for 40- electronic spectra but quite distinct heme properties. Kinetic 60% of the total copper. The ENDOR data indicate the presence of experiments for binding of O2, CO and NO were carried out by an exchangeable solvent ligand associated with the type 2 Cu (II) flash-photolysis, along with Kd measurements. DosT and DevS signal. Various substrates and products were incubated with pMMO autophosphorylation reactions were clearly inhibited by O2 while from M. capsulatus (Bath) to probe the active site. NO and CO did not alter the initial rates. Our results show DosT and DevS are oxygen sensors and explain why NO was mistakenly assumed as signal. We present our working model for this system. P179 Spectroscopic insight into the Fe(II) environment at the P181 ferroxidase site in Ferritins Jennifer K. Schwartz1, Xiaofeng S. Liu2,3, Elizabeth C. Theil2,3, Heme to protein ester linkages in mammalian Edward I. Solomon1, 1Stanford University, Stanford, CA, USA; peroxidases 2CHORI (Children’s Hospital Oakland Research Institute), Johanna M. Stampler, Martina Zederbauer, Paul G. Furtmüller, Oakland, CA, USA; 3University of California-Berkeley, Berkeley, Department of Chemistry, Division of Biochemistry, Metalloprotein CA, USA. Research Group, 1190 Wien, Austria. Contact e-mail: [email protected] Contact e-mail: [email protected]

Ferritin, an essential protein in humans, plants, bacteria, and other In mammalian peroxidases the heme is covalently attached to the animals, is able to concentrate large amounts of iron as hydrated protein via two ester linkages between the carboxyl groups of ferric oxides for iron-protein synthesis and minimize free radical conserved glutamate and aspartate residues. This interaction of the generation with dioxygen/peroxide. Relatively little is known about protein matrix with the chromophore is responsible for the the electronic and geometric structure of the biferrous oxidase sites extraordinary spectral, redox and enzymatic features of these that form ferric mineral precursors in maxi-ferritins, 24 subunit metalloproteins. In the present study, recombinant MPO and the protein nanocages, or mini-ferritins (Dps, DNA protection during variants Asp94Asn, Asp94Val and Glu242Gln were investigated in starvation, proteins), 12- subunit protein nanocages. Maxi-ferritin a comparative biophysical and biochemical study. We report the ferroxidase site ligands for bi-iron substrates are A: E, ExxH; B: E, consequences of mutations on the spectral features, redox QxxD/S/A (Liu and Theil, PNAS 101: 8557, 2004). We have thermodynamics of the ferric/ferrous couple and inter-conversion of studied the ferroxidase sites of frog m-ferritin (mFr) maxi-ferritin redox intermediates of the halogenation cycle. Disruption of the and paired Bacilli, Dps 1 and 2 mini-ferritins, with a combination of Asp ester bond had no effect on the kinetics of binding of low spin circular dichroism (CD), magnetic circular dichroism (MCD), and ligands (cyanide and nitrite) as well as on the formation of variable-temperature, variable-field MCD (VTVH MCD) to probe compound I mediated by either hydrogen peroxide or hypochlorous Fe (II) binding to the active sites, as well as their excited and acid. Additionally, its impact on the reduction of compound I by ground states reflecting their coordination environment and iodide and thiocyanate was negligible. By contrast, rates of bridging ligation. CD spectra of the paired Bacilli Dps mini-ferritins compound I reduction by chloride and bromide were substantially indicate similar protein environments, however large differences in decreased compared with the wild-type protein. Disruption of the the MCD and VTVH MCD show changes in the geometry and Glu ester bond significantly increased heme flexibility, blue-shifted ground states of the maxi versus mini ferritin ferroxidase sites. the UV-Vis spectrum decelerated both cyanide binding and Oxidant preferences in maxi-and mini-ferritins also differ: dioxygen compound I formation. The apparent bimolecular rate constants at 123 S92 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

pH 7.0 of compound I reduction by chloride, bromide, iodide and epoxidation and allylic C-H hydroxylation of cyclohexene. Herein, thiocyanate were 500-, 25-, 21- and 63-times decreased compared we reveal that entropy of activation, ǻS‡, is more important with recombinant wild-type MPO. The effects of exchange of thermodynamic factor to control the chemoselectivity than Asp94 and Glu242 on electron transfer reactions are discussed with activation energy, ǻEa. This result clearly explains drastic respect to the known structures of MPO and the MPO-cyanide and temperature effect on chemoselectivity. In additional, the present MPO-halide complexes. result suggests re-consideration of the discussion based on ǻEa, calculated by the density functional theory (DFT). P182 Valence delocalization in the [2Fe-2S]+ centers of mutant forms of Aquifex aeolicus Ferredoxin 4 Sowmya Subramanian1, Jacques Meyer2, Michael K. Johnson1, 1Department of Chemistry and Center for Metalloenzyme Stuides, University of Georgia, Athens, GA, USA; 2Département Réponse et Dynamique Cellularies, CEA-Grenoble, Grenoble, France. Contact e-mail: [email protected]

Aquifex aeolicus Ferredoxin 4 (AaeFd4) is the best structurally characterized member of the class of the thioredoxin-like [2Fe-2S] Fds. High-resolution crystal structures are available for the wild- type (WT) protein as well as the C55S and C59S variants with Fe- S/Fe-O/Fe-Fe distances accurate to ±0.01 [[Unable to Display P184 Character: Ǻ]] (1). The reduced forms of the corresponding "Effector" roles of the electron donor protein in C56S and C60S variants of the closely related Clostridium pasteurianum [2Fe-2S] Fd are currently the only known samples of P450cam-catalyzed oxygenation reaction valence-delocalized [2Fe-2S]+ clusters (2). Such cluster fragments Kenta Tanemura, Takeshi Uchida, Koichiro Ishimori, Division of constitute a fundamental building block of all higher nuclearity Fe- Chemistry, Graduate School of Science, Hokkaido University, S clusters and understanding the origin of valence-delocalization is Sapporo, Japan. central to the understanding of biophysical properties of Fe-S Contact e-mail: [email protected] clusters in general. In this work, the spectroscopic properties of the P450cam is a heme-containing enzyme that catalyzes the [2Fe-2S]2+,+ center in WT and the C55S, C59S, C55S/R13E and hydroxylation of D-camphor by using dioxygen and two electrons. C59S/R13E mutant forms of AaeFd4 have been studied by UV- To accept electrons from the redox partner, Pdx, the specific Visible absorption, EPR, resonance Raman, and variable structural changes by binding of Pdx are required in P450cam, temperature MCD spectroscopies in an effort to understand the indicating that the Pdx has ‘effector’ functions for the catalytic origin of valence delocalization. Valence delocalized species with cycle of P450cam. Our previous study suggested that the steric ferromagnetically coupled S = 9/2 ground states were found to exist interaction mediated by the side chain of Leu-358 plays a key role as minority species in frozen samples of each of the dithionite- in the propagation of the structural change from the Pdx binding site reduced AaeFd4 mutants at pH 11. However, variable temperature to the heme moiety, but details of this interaction are still unclear. In UV-visible absorption and MCD studies at pH 11 indicate that the this study, we newly prepared a mutant, L358A, in which Ala is mutants exclusively contain valence-delocalized [2Fe-2S]+ centers introduced into the position of 358 to perturb the steric interaction with S = 9/2 ground states at room temperature and only become with heme, and examined the heme environmental structure by valence-localized with antiferromagnetically coupled S = 1/2 using the resonance Raman spectroscopy. The Fe-C stretching ( ground states on freezing. The origin of valence-delocalization in ȞFe- ) and C-O stretching ( ) modes in carbonmonoxy L358A, [2Fe-2S]+ centers will be discussed in light of these results. CO ȞC-O which serve as the sensitive markers for the heme environment, were observed at 481 cm-1 and 1940 cm-1, respectively. In addition, -1 the binding of Pdx to L358A upshifted ȞFe-CO by 2 cm and P183 -1 downshifted ȞC-O by 8 cm as found for wild type P450cam. Based A Thermodynamic Analysis of Epoxidation versus Allylic on these results, we can conclude that the L358-mediated steric C-H Hydroxylation on Cyclohexene Oxygenation by interaction would not be so crucial for the effector functions of Pdx, High-Valent Iron-Porphyrin Complexes Related to which allows us to speculate that the Pdx binding induces the Cytochrome P450 conformational change of the main chain rather than the side chain Akihiro Takahashi, Takuya Kurahashi, Hiroshi Fujii, Institute for of Leu358 which enables P450cam to accept electrons for the Molecular Science and Okazaki Institute for Integrative Bioscience, catalytic reaction. National Institutes of Natural Sciences, The Graduate University for Advanced Studies, Okazaki, Japan. Contact e-mail: [email protected] P185 Tryptophan 2,3-dioxygenase; structural and mechanistic It has been recognized that oxoiron(IV) porphyrin ʌ-cation radical studies species (the so-called compound I) is involved as a reactive Sarah J. Thackray, Ross J. L. Anderson, Chris Mowat, Chiara intermediate in catalytic cycles of cytochrome P450, peroxidase, Bruckmann, Stephen K. Chapman, University of Edinburgh, and catalase. The compound I in Cytochrome P450 can catalyze Edinburgh, United Kingdom. alkene epoxidation and hydrocarbon hydroxylation. Interestingly, Contact e-mail: [email protected] cyclohexene, which contains the C=C and allylic C-H functionalities in one molecular, is converted to cyclohexene oxide Tryptophan 2,3-dioxygenase (TDO) is a heme-containing enzyme (epoxidation product) and cyclohexen-3-ol (hydroxylation product) from a family of enzymes including indoleamine 2,3-dioxygenase by the compound I in cytochrome P450. It is reported that the (IDO). TDO is a homotetrameric enzyme and has a high specificity chemoselectivity of epoxidation versus allylic hydroxylation of for L-tryptophan (L-Trp) and related derivatives such as 6-F-Trp. cyclohexene oxygenation are controlled by various factors such as IDO is monomeric, and shows activity toward a range of substrates, reaction temperature, solvent, electronic nature and axial ligand of including L-Trp, D-tryptophan and tryptamine. TDO and IDO have iron porphyrin. At present, however, it remain unclear how these been implicated in a number of physiological conditions, including factors control the chemoselectivity of cyclohexen oxygenation. In this study, we carried out that detailed kinetic analysis of C=C 123 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98 S93

suppression of T cell proliferation and the immune escape of P187 cancers, making them attractive targets for drug discovery. Ferritin-specific di-iron site ligands control rapid iron To provide insight into substrate recognition and catalysis we have mineralization pathways Xanthomonus campestris solved the structure of TDO from , Takehiko Tosha1, Elizabeth C. Theil1,2, 1Council on Bioiron at L complexed with -Trp and 6-F-Trp, to a resolution of 1.6Å and Children's Hosp. Oakland Res. Inst., Oakland, CA, USA; 2Dept. 1.9Å respectively. Both are in the catalytically active, ferrous heme Nutri. Sci. & Toxicol., UC-Berkeley, Berkeley, CA, USA. state. These show that TDO is an induced-fit enzyme with Contact e-mail: [email protected] significant structural changes on binding of substrate. They also define the structural basis for the stereospecificity of TDO. It is also Ferritin initiates iron mineralization by catalytic coupling of ferrous L- shown that a Trp residue binds to an allosteric site at the interface iron and molecular oxygen at multiple, di-iron sites A and B between each monomer, offering a possible effector site for containing some of ligands common to diiron oxygenases where L- allosteric activation of TDO by Trp. iron is a cofactor, in contrast to ferritin where iron is a substrate. Biochemical studies also provide direct evidence for the induced-fit Site A, Glu, Glu-x-x-His is common to all the di-iron proteins (eg. behaviour - electrochemical and kinetic studies show that there is a methane monooxygenase, ribonucleotide reductase, ferritins) but significant stabilization of the ferrous form when substrate is bound. site B, Glu, Gln-x-x-Asp/Ser/Ala is ferritin-specific.. Here we report This stabilization could play a physiological role keeping the the catalytic effects of engineering ferritin site B to mimic the protein reduced, and therefore active. oxygenase sites with the following changes: Site B: EED- Gln137Glu, site B-EQH-Asp140His, and site B-EEH- Gln137Glu/Asp140His). Substitution of His for Asp, whether in B site EQH or EEH mutant, inactivated catalysis with the initial rates of the ferrous oxidation decreased to 1.0-2.0% of WT ferritin. However, for B-site EED, with the conservative Gln137Glu substitution, catalysis was only slightly inhibited (73% of that of WT). In spite of the similar initial rates, the reaction pathway was altered by the Gln/Glu mutation in Site B-EED since the di-ferric peroxo intermediate was undetectable and, using entry into the slower phases of mineral formation as a reporter, dissociation of the diferric oxy (hydroxyo) product and mineral precursor appeared to be inhibited. These results indicate that ferritin-specific Site B iron ligands control the reaction pathway for rapid iron oxidation/coupling and release of diferric oxo/hydroxo mineral precursors in rapid iron mineralization of ferritin. Part support DK20251 (ECT and TT) and the JSPS (TT).

P188 Heme Transfer Mechanism from Heme ChaperonE, CcmE, to Apocytochrome c Takeshi Uchida1, Yuka Kondo1, Ayako Tamura1, Julie M. P186 Stevens2, Stuart J. Ferguson2, Koichiro Ishimori1, 1Hokkaido 2 The catalytic center of a Desaturase from Arabidopsis University, Sapporo, Japan; Department of Biochemistry, thaliana University of Oxford, Oxford, United Kingdom. Contact e-mail: [email protected] Cristina G. Timoteo, Cristina Pantana, Américo G. Duarte, Filipe Folgosa, Alice S. Pereira, Pedro Tavares, Requimte, Centro de c-type cytochromes are electron transfer proteins that are essential Química Fina e Biotecnologia, Caparica, Portugal. for the life of virtually all organisms. They characteristically carry Contact e-mail: [email protected] covalently-bound heme via thioether bonds to two cysteines in the protein. In Gram-negative bacteria, biogenesis of c-type cytochrome Fat acid synthesis is a complex process which involves many is conducted by a multiprotein complex system known as a catalytic steps. Unsaturated fat acids are obtained through a cytochrome c maturation (Ccm) system. This system consists of 8 desaturation reaction, a process in which a single bond between two gene products (ccmA-ccmH). CcmE, which binds heme and delivers carbon atoms is converted in a double bond. This can be an oxygen it to apocytochrome c, is called as a heme chaperonE. In the dependent process, catalyzed by an enzyme named desaturase. previous study, covalent attachment of heme to cytochrome c was From an evolutionary point of view, there are two classes of achieved in the presence of only CcmE, heme, and dithiothreitol. desaturases: soluble and membrane bound. Both classes are capable 9 However, the precise mechanism remains to be clarified. In this of ǻ -desaturation of the acyl 18:0 chains, and require two electrons study, we applied UV/vis absorption and resonance Raman for each double bond formed. The presence of an iron centre in the spectroscopies to the reaction of CcmE with apocytochrome c to soluble desaturases was revealed for the first time through understand the mechanism of heme transfer and covalent bond Mössbauer spectroscopy. Previous studies indicate that this centre is formation. absent in the membrane bound enzymes. After addition of apocytochrome c into CcmE in the presence of A membrane bound desaturase from Arabidopsis thaliana, was E.coli sodium dithionite and dithiothreitol, the Soret maximum shifted to cloned and overexpressed in BL21 (DE3) cells. Biochemical 417 nm from 400 nm, and two new bands appeared at 527 and 533 and spectroscopic studies will be presented which establish the nm. The positions of these bands were different from those of presence of an iron centre in this enzyme. neither CcmE nor cytochrome c in the reduced form. Unfortunately, resonance Raman spectrum of the mixture of CcmE and apocytochrome c under the same condition was hindered by strong fluorescence probably caused by the dissociated heme. The results of the absorption spectral changes suggest that heme is not transferred from CcmE to apocytochrome c simultaneously, but an unspecified transient form is formed during the heme transfer.

123 S94 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

P189 P191 Crystal structures of the hydroperoxo-heme- and Į-meso- Looking for peroxidase substrates and binding site(s) in hydroxyheme intermediates in heme oxygenase catalysis bifunctional catalase peroxidases Masaki Unno, Masao Ikeda-Saito, Tohoku University, Sendai, Jutta Vlasits, Marcel Zamocky, Christa Jakopitsch, Christian Japan. Obinger, University of Natural Resources and Applied Life Contact e-mail: [email protected] Sciences, Wien, Austria. Contact e-mail: [email protected] Heme oxygenase (HO) catalyzes the O2-dependent conversion of heme to biliverdin, Fe, and CO through three successive Catalase-peroxidases (KatGs) exhibit peroxidase and substantial oxygenation steps in which the heme participates both as a substrate catalase activities similar to monofunctional . Crystal and as a cofactor. The first oxygenation of heme occurs structures of four different prokaryotic KatGs reveal the presence of regiospecifically at the Į-meso position to afford Į-meso- a peroxidase-conserved proximal and distal heme pocket together hydroxyheme. A ferric hydroperoxo intermediate (FeIII-OOH) has with features unique to KatG. Marked differences in the structural been identified as the meso-hydroxylating species, different from role of conserved amino acids and hydrogen-bond networks in other heme enzymes which employ compound I as their reactive KatG with respect to other plant-type heme peroxidases were species. To gain further insight into the HO catalytic mechanism, found.* Typically, the catalatic but not the peroxidatic activity was we determined the crystal structures of the hydroperoxo heme- and very sensitive to mutations that disrupted the KatG-typical the Į-meso-hydroxyheme intermediates of HmuO, a bacterial HO, extensive hydrogen-bonding network.* By contrast, the endogenous at 1.9 Å and 1.5 Å resolutions, respectively. The hydroperoxo peroxidase substrate(s) and binding site(s) in KatG are unknown. intermediate was generated by X-ray-induced photoreduction of the Typical for KatG is a unique distal Trp-Tyr-Met adduct with a oxy crystal at cryogenic temperature. This intermediate was highly joining arginine that can move between two conformations. The susceptible to the incident X-ray radiation, and only merging the side-chain of this Arg is located on the surface of a cleft with a independent diffraction data from 20 crystals attained successful well-defined U-shaped region. Structural analysis and sequence structure determination. The Fe-O distance is slightly longer and alignment revealed the presence of several highly conserved Fe-O-O angle is smaller by 5 ° than those found in the oxy residues in this region that could play a role in binding and structure. The diffraction data set for the hydroxyheme intermediate oxidation of one-electron donors. Here, we present data on was collected after brief annealing the crystal of the hydroperoxo Synechocystis KatG and the effect of exchange of R509, W459, intermediate near room temperature. The hydroxyheme shifts along K435, N278 and E281 on the bifunctional activity and the heme Į-Ȗ axis, and the distal helix moves in the opposite interconversion and spectral properties of redox intermediates. direction compared with the hydroperoxo heme complex. The *Smulevich, G., Jakopitsch, G., Droghetti, E., Obinger, C. (2006) proton, required for activation of hydroperoxo, could be delivered Probing the structure and bifunctionality of catalase-peroxidase to the proximal oxygen from nearby water through this (KatG). J. Inorg. Biochem. 100, 568-585 rearrangement of the active site. P192 P190 Magnetic circular dichroism of Aminopeptidase from Metal-free SOD1 forms amyloid-like oligomers: a Aeromonas proteolytica possible general mechanism for familial ALS Adam S. Volwiler, James A. Larrabee, Middlebury College, Miguela Vieru1, Lucia Banci1, Ivano Bertini1, Stefania Girotto1, Middlebury, VT, USA. Manuele Martinelli1, Julian P. Whitelegge2, Armando Durazo2, Joan Contact e-mail: [email protected] Selverstone Valentine2, 1Magnetic Resonance Center, Florence, Italy; 2University of California, Los Angeles, CA, USA. Aminopeptidase from Aeromonas proteolytica (AAP) serves as an Contact e-mail: [email protected] example of a metallohydrolase in which the active site structure has a ȝ-hydroxo-ȝ-carboxylato-dimetal core. This core is a common Amyotrophic lateral sclerosis (ALS) is a progressive degenerative structural motif in many metallohydrolases which are active in the disease of motor neurons. The inherited form of the disease, familial dicobalt(II) forms including methionine aminopeptidase (MetAP) ALS, represents almost 10% of the cases reported and there is and glycerophosphodiesterase (GpdQ). One interesting feature of strong evidence that these are due to the mutations in the gene the dicobalt(II) active site is the extent of magnetic exchange encoding copper-zinc superoxide dismutase (SOD1). Recent studies coupling between the Co(II) ions. Magnetic circular dichroism showed that SOD1 mutations, more than the loss of normal (MCD) can be used to determine magnetic exchange coupling, and function, induce gain of a toxic function. The finding of the AAP active site is particularly well suited for MCD study proteinaceous cytoplasmic inclusions containing SOD1 in motor because three different metal-substituted forms, [CoZn(AAP)], neurons of postmortem familial ALS patients, suggests that [ZnCo(AAP)], and [CoCo(AAP)], can be prepared. Any magnetic aggregation of mutant SOD1 produces a pathogenic species, and the exchange coupling between Co(II) ions is turned off in either the insoluble aggregates may represent only the final form of a previous [CoZn(AAP)] or [ZnCo(AAP)] form. Furthermore the pathological state, which is eliminated by cells inducing its spectroscopic properties and electronic ground state of each Co(II) aggregation.We have characterized the WT SOD1 protein in the apo ion can separately probed, and changes in the ground state and metallated states with respect to its ability to form aggregates. properties in the [CoCo(AAP)] form can be attributed to exchange Fluorescence studies using the Thioflavin T probe, coupled with gel coupling. The study of AAP provides a basis for the more general chromatography, show that a soluble aggregate forms in vitro understanding of all dinuclear metallohydrolases. during incubation at 37 ºC of an apo sample of SOD1 at physiological conditions (pH 7, 100 ȝM). We have further characterized this aggregate. This species might constitute the first seed for a subsequent insoluble aggregate formation. Further studies will focus on investigating the propensity of a selected set of pathogenic SOD1 mutants to form soluble aggregates.

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P193 P195 Flavo-Diiron Enzymes: Nitric Oxide and/or Dioxygen Regulation of myoglobin functions by self-aggregates of Reductases? an anionic porphyrin David A. Wampler1, Angela- Nadia Albetel2, Donald M. Kurtz, Kenji Watanabe, Yoshiyuki Ishida, Koji Kano, Doshisha Jr.1, 1Department of Chemistry, University of Texas at San Antonio, University, Kyotanabe, Kyoto, Japan. San Antonio, TX, USA; 2Department of Chemistry, University of Contact e-mail: [email protected] Georgia, Athens, GA, USA. Contact e-mail: [email protected] In chemical biology, modification of protein functionality is one of the major subjects. Synthetic molecules that complex with a specific A widespread group of bacterial and archaeal flavo-diiron enzymes protein and alter a conformation of the protein have a potential to (FprAs) contain an active site consisting of a flavin juxtaposed with regulate the functions of the target protein. We studied a non-heme His,Glu,Asp-ligated diiron site. FprAs have no complexation of self-aggregates of 5,10,15,20-tetrakis(4- detectable amino acid sequence homology to any other class of non- sulfonatophenyl)porphyrin (TPPS) with metmyoglobin (metMb) heme iron proteins. Some FprAs clearly function as scavenging leading to change in the secondary structure of metMb and increase nitric oxide reductases (S-NORs) under anaerobic growth in the binding affinity of metMb for the azide anion. TPPS is known conditions. In vitro, however, some FprAs are reported to have to form extended J-aggregates in an aqueous acidic solution. dioxygen reductase (O2R) activity, either instead of or in addition However, under neutral conditions, TPPS exists as the monomer to NOR activity. We are undertaking a systematic survey of these and/or the dimmer. In this study, we found that J-aggregates of two activities, both in vivo and in vitro, of FprAs from various TPPS can exist stably for several hours even in an aqueous neutral bacteria and archaea. Attempts to correlate the relative levels of solution when the aggregates are bound to metMb (Figure 1). these two activities with spectroscopic and structural differences Circular dichroism study indicates that binding of the J-aggregates among the FprAs will be described, as will progress in leads to a change in the secondary structure of metMb. The binding understanding the catalytic mechanisms. constants (K) for association of the azide anion to metMb in the absence and the presence of the J-aggregates as well as the monomer of TPPS were determined. The results revealed that the J- P194 aggregates enhance the binding of the azide anion to metMb, while Typical and untypical metal ion binding modes in Cicer the TPPS monomer does not show significant effect (Figure 2). arietinum metallothionein 2 Xiaoqiong Wan, Eva Freisinger, Institute of Inorganic Chemistry, Zürich, Switzerland. Contact e-mail: [email protected]

Metallothioneins (MTs) are small cysteine-rich metal ion binding proteins found in a huge number of species. Nevertheless, only few attempts have been performed to study MTs in plants until now leaving the knowledge about their functions and the related mechanisms still mostly in the dark. C. arietinum (chickpea) MT2 shows the characteristic cysteine distribution pattern of the p2 subfamily of plant MTs [1] including the high affinity to d10 metal ions typical for all MTs. We will present the metal ion binding affinities of MT2 to essential or nonessential metal ions, e.g. ZnII, CdII , HgII, CuI and CoII, which were studied using a plethora of spectroscopic as well as (bio-) chemical methods. Specifically, changes in coordination geometry upon stepwise addition of the different metal ions will be shown. We were further able to identify an additional metal ion binding site with weaker affinity, which seems to be independent of the metal-thiolate clusters formed. Financial support for this project comes from the Swiss National Science Foundation (200020-113728/1 to EF). [1] Y. Kojima, P.-A. Binz, J.H.R. Kägi, Nomenclature of metallothionein: proposal for a revision, in: C. Klaassen (Ed.), Metallothionein IV, Birkhäuser Verlag, Basel, 1999, p. 3.

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P196 Crystal structure of histidine kinase and response regulator complex: insight into regulatory mechanism of oxygen sensor FixL Seiji Yamada1, Miki Kobayashi1,2, Hiroshi Sugimoto1, Hiro Nakamura1,3, Yoshitsugu Shiro1, 1RIKEN/SPring-8 Center, Sayo, Hyogo, Japan; 2University of Hyogo, Ako, Hyogo, Japan; 3Yokohama City University, Yokohama, Kanagawa, Japan. Contact e-mail: [email protected]

Rhizobia have exploited the oxygen sensing two-component system, FixL/FixJ (histidine kinase/response regulator), to express the nitrogen fixation enzymes under anaerobic conditions. FixL is a heme-containing histidine kinase, and is divided into sensor, dimerization, and catalytic domains. Under anaerobic conditions, FixL phosphorylates own histidine residue by using ATP. The phosphoryl group is subsequently transferred to the conserved aspartate residue in FixJ. However, the kinase reaction is inhibited in high oxygen concentrations. Structural analyses of sensor domain P198 of FixL revealed the intra-domain conformational change upon Inhibition of the SARS coronavirus by bismuth oxygen binding. However, it is still unknown how the signal is compounds transferred to the downstream domains, because no full-length 1 2 2 3 Nan Yang , Julian A. Tanner , Jian-Dong Huang , Bojian Zheng , structure of FixL is available. 1 1 Hongzhe Sun , Department of Chemistry, The University of Hong Then, we determined the crystal structure of the structural homolog 2 Kong, Hong Kong, China; Department of Biochemistry, The of FixL/FixJ, ThkA/TrrA from hyperthermophile. The structures of 3 University of Hong Kong, Hong Kong, China; Department of sensor, dimerization, and catalytic domains and TrrA were Microbiology, The University of Hong Kong, Hong Kong, China. determined at 1.6-1.9 Å resolution. The determined structures were Contact e-mail: [email protected] fitted into the ThkA/TrrA complex obtained at 3.7 Å resolution. The determined structure revealed new insights as follows: (1) the inter- As one of the widely used elements in clinic for centuries,[1] the domain interaction between sensor and catalytic domain regulates application of bismuth-based drugs was extended in medical and the kinase activity, (2) the ATP binding site of catalytic domain health purposes, e.g., anticancer, antimicrobial, and antivirus.[2,3] changes the conformation after the kinase reaction, and (3) the inter- SARS-CoV (SCV) was first recognized in late 2002 and its main domain interaction between sensor and FixJ regulates the ligand proteinase, cysteine proteinases, NTPase/helicase and RNA affinity in the sensor domain. polymerase have been identified as potential anti-viral targets, and have already undergone preliminary characterization.[4] Our P197 research found that bismuth complexes exhibit effective inhibition to SCV helicase both ATPase and duplex-unwinding activities, Dynamic roles of specific heme-ligand-residue probably due to the binding of Bi3+ to the metal binding domain, a interactions in the oxygen sensor proteins Dos and FixL Cys-rich and Zn2+-bound region in the N-terminus of the enzyme. Taku Yamashita, Latifa Bouzhir-Sima, Ursula Liebl, Marten H. Our cell culture experiments also show the inhibition of Bi3+ to Vos, INSERM U696 - CNRS 7645, Ecole Polytechnique, Palaiseau, SCV, revealing that Bi3+ plays the inhibitory role during later stages France. of virus replicative cycle. Contact e-mail: [email protected] Keywords: bismuth, SARS, helicase

FixL and Dos are bacterial heme-based oxygen sensor proteins, in which binding/release of O2 to/from the heme in a heme-binding PAS-domain leads to changes in the activity of an associated enzymatic (kinase or phosphodiesterase) domain. The structures of the PAS domains FixLH (red) and DosH (blue) have been solved and are generally similar (Figure), but the heme coordination is markedly different: the unliganded heme is 5-coordinated in FixLH, like in myoglobin, but in DosH O2 replaces an internal ligand (Met 95 in E. coli). Heme-ligand photodissociation can in principle be used to trigger ‘switching’ of the sensor and identify intermediates in the intra-protein signaling pathway. Ultrafast spectroscopic References: . experiments indicate that unusual heme-ligand interactions are [1] (a) Sun H, Zhang L, Szeto K (2004) Metal Ions Biol. Syst 41: maintained after dissociation and that the heme pocket acts as an 333. (b) Yang N, Sun H (2007) Coord. Chem. Rev., in press. et al. oxygen trap (1,2). With the aim of exploring the role of specific [2] Qu CF (2005) Cancer Biol. Ther. 4: 849. et al. heme-ligand-residue interactions in functional motions, we have [3] Magnusson NE (2005) Cell Tissue Res. 321: 195. et al. constructed mutants proteins (in particular with modified Met 95 in [4] (a) Tanner, JA (2003) J. Biol. Chem. 278: 39578. (b) t al. DosH and Arg 206 in FixLH from B. japonicum, that interacts with Anand K e (2003) Science 300: 1763. the propionates), and observed substantial changes in early heme- ligand interactions. Interpretations are aided by molecular modeling. 1. Liebl, U., Bouzhir-Sima, L., Négrerie, M., Martin, J.-L., & Vos, M. H., (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 12771-12776. 2. Jasaitis, A., Hola, K., Bouzhir-Sima, L., Lambry, J.-C., Balland, V., Vos, M. H., and Liebl, U., (2006) Biochemistry 45, 6018-6026.

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P199 06/01/0004, CARM, Murcia; as well as Project BIO2006-15363, Fungal catalase-peroxidases - a novel group of MEC, Madrid. (R.M.G.) NOVOZYMES SPAIN S.A. (R.Z.) bifunctional oxidoreductases Doctorate fellowship AP-2004-5759, FPU-MEC, Madrid. Marcel Zamocky, Christa Jakopitsch, Jutta Vlasits, Christian Obinger, Division of Biochemistry, Department of Chemistry BOKU, Wien, Austria. P201 Contact e-mail: [email protected] Tryptophan-to-dye fluorescence energy transfer applied to oxygen sensing using type-3 copper proteins. Catalase-peroxidases (KatGs) are bifunctional heme b containing Gerhild Zauner1, Thijs J. Aartsma2, Gerard W. Canters1, Armand 1 1 oxidoreductases exhibiting catalase (2 H2O2 ---> 2 H2O + O2) and W. J. W, Tepper , Leiden Institute of Chemistry, Leiden, The • 2 peroxidase activity (H2O2 + 2 AH2 --> 2 H2O + 2 AH). All known Netherlands; Leiden Institute of Biophysics, Leiden, The catalase-peroxidases belong to class I of the superfamily of Netherlands. bacterial, fungal, and plant heme peroxidases. Four crystal Contact e-mail: [email protected] structures of prokaryotic KatGs are known revealing several peculiar structural features. Sequence similarity searches and A fluorescence-based system to sense oxygen is described. The phylogenetic analysis of 134 eukaryotic genomes revealed the method exploits the sensitivity of the fluorescence of type-3 copper presence of 23 fungal and 3 protist katG genes that form a novel proteins towards oxygen by translating the near-UV emission of the and distinct group within the abundant catalase-peroxidase protein protein to label fluorescence in the visible range through a FRET family. Mainly in the catalytic domain their sequence similarity mechanism (Figure 1). The main protein in this study, a with bacterial KatGs is rather high but multiple sequence recombinant tyrosinase from the soil bacterium Streptomyces alignments clearly suggest differences between prokaryotic and antibioticus, has been covalently labeled with a variety of eukaryotic proteins. fluorescent dye molecules with emission maxima spanning the In phytopathogenic fungi KatGs are probably involved in host- visible wavelength range. The emission of the label varied pathogen interactions. Thus, investigations of fungal KatGs have a considerably between O2-bound and O2-free protein with a contrast great practical impact for developing tools that prevent plant exceeding that of the Trp emission for some labels. Different affection by these pathogens.Here, we focus on the cloning and constructs may be simultaneously observed using a single excitation heterologous expression of catalase-peroxidases from the two wavelength. Next to oxygen sensing, the method may be applicable phytopathogenic fungi Gibberella zeae and Magnaporthe grisea to any protein showing variations in tryptophan fluorescence, for both having two katG paralogs. In the case of Gibberella zeae example as a function of ligand binding or catalysis. katG1 a rare fusion of katG gene with a region coding for a cytochrome P450-like domain was detected thus forming a putative unique trifunctional multidomain heme containing oxidoreductase. This research is supported by the Austrian Science Foundation (FWF) project number P19793_B11.

P200 Peroxidase affinity towards hydrogen peroxide: Determination from a conversion time method. Rosa Zamora1, Virginia Tomas2, Ramiro Martinez-Gutierrez3, Francisco Garcia-Canovas1, Jose Tudela4, 1GENZ-Grupo de investigacion Enzimologia, Universidad de Murcia, Spain; 2Dept. Quimica Analitica, Universidad de Murcia, Spain; 3NOVOZYMES Spain S.A., Madrid, Spain; 4GENZ (http://www.um.es/genz), Universidad de Murcia, Spain. Contact e-mail: [email protected]

Peroxidase (POD, EC 1.11.1.7) is located in humans, animals, plants and microorganisms, and it is involved in a number of peroxidative processes with biotechnological interest. The enzyme catalyzes the oxidation by hydrogen peroxide of reductant substrates to radicals, which decompose by means of non-enzymatic reactions up to polymers. POD has great affinity towards hydrogen peroxide, thus it may be difficult to determine the value of its Michaelis constant using steady state rate assays. An alternative method consists of the use of progress curves of assays with hydrogen peroxide depletion. Figure 1. Structure of Streptomyces antibioticus tyrosinase (left), The analysis of the data points of a progress curve needs its modelled after [PDB 1WX3] showing the type-3 Cu atoms, the complex fitting by non-linear regression to an implicit equation, the coordinating His residues, the 12 Trp residues and Thr1, integrated equation of Michaelis. A simpler method is based on the corresponding to the attachment point of the label. The principle of derivation of the analytical expressions of the conversion time. the FRET based O2 sensing is shown at the right. There have been obtained linear and hyperbolic useful expressions for the determination of Michaelis constant. Besides, there have been established equations that allow predicting the duration of the reactions of substrate depletion. The method has been applied successfully for the determination of the Michaelis constants of POD towards hydrogen peroxide and 4-tertbuthylcatechol as substrates. The authors are grateful to NOVOZYMES SPAIN S.A. by free samples of peroxidase. This work has been partially supported by grants from several Spanish organizations. (J.T.) Projects 00672/PI/04 Fundacion Seneca and BioCARM BIO-BMC 123 S98 J Biol Inorg Chem (2007) 12 (Suppl 1):S53–S98

P202 Overexpression and characterization of a histidine- and glutamine-rich protein, Hpn-like Yibo Zeng, Dongmei Zhang, Hongzhe Sun, Department of Chemistry and Open Laboratory of Chemical Biology,The University of Hong Kong, Hong Kong, China. Contact e-mail: [email protected]

Helicobacter pylori is a micro-aerophilic, Gram-negative, spiral- shaped organism. [1-2] Similar to Hpn, Hpn-like (Hpnl), a histidine- and glutamine-rich (25% and 40%) protein in H. pylori, may play a putative role in nickel detoxification and influence the active levels of Ni-activated urease.[3-4] We cloned gene from H. pylori 11637 strains, over-expressed in E. coli BL21(DE3), and purified the protein. Our UV-vis spectra reveals Hpnl binds 2 nickel ions per monomer at pH 7.5. This binding was reversible: metal could be released from Hpnl in the presence of the chelating ligand EDTA. Ni2+ also induced conformation changes of the protein, with Į-helix content decrease from 26% to 20% and ȕ-sheet increase from 14% to 22%. Growth curves of E. coli BL21 both with and without hpnl gene implied a role for Hpnl to protect the cells from higher concentrations of outside Ni2+. Therefore Hpnl may serve multiple roles inside the bacterium: storage of Ni2+ ions; donation Ni2+ to metalloenzymes or other proteins; and detoxification via sequestration of excess Ni2+. Keywords: Helicobacter pylori, Hpnl Acknowledgement: This work was supported by Area of Excellence of UGC and URC of the University of Hong Kong! References: 1. Mulrooney SB, Hausinger RP (2003) FEMS Microbiol Rev 27: 451-480. 2. Dosanjh NS, Michel SL (2006) Curr Opin Chem Biol 10: 123- 130. 3. Ge R, et al. (2006) Biochem J 393: 285-93. 4. Ge R, et al. (2006) J Am Chem Soc 128: 11330-11331.

P203 Molecular characterisation of metal transporters in the plant Arabidopsis Thaliana Matthias Zimmermann, Zhiguang Xiao, Anthony Gordon Wedd, School of Chemistry and Bio 21 Institute, The University of Melbourne, Parkville, Australia. Contact e-mail: [email protected]

Fourteen metals are known to be essential for plants and animals. However, their concentrations in cells have to be strictly regulated at their optimum levels, since too much or too little is often lethal. The molecular details of these control mechanisms are poorly understood in most cases. The simple plant Arabidopsis thaliana has eight P1B-type ATPase (HMA 1-8) responsible for uptake and elimination of various metals. Phylogenetic analysis shows that HMA 1-4 are likely to transport divalent cations and that HMA 5-8 are likely to transport monovalent cations. However, the molecular basis of such metal transporting specificity is not known. The N-terminus of each HMA protein, HMAn comprises one or more metal binding domains that may contribute to the metal specificity. For example, a metal binding motif (CXXC; C, cysteine; X, another amino acid) typical for Cu(I) was present in HMA7n while a cysteine pair (CC) was found in both HMA2n and HMA4n and may promote Zn(II) binding. To understand the possible roles of the N-terminus in metal binding and selection, we have cloned, expressed and purified the N-terminal domains of the HMA4 and HMA7 with selected mutants. Metal binding affinity and specificity have been studied, together with structural aspects. The results will be presented and discussed.

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